Immunoassays in capillary tubes

ABSTRACT

A fluorescent immunoassay employing the interior surface of a capillary tube is provided. Devices to permit immunoassays using one or more capillary tubes, an apparatus for use with the devices, and a process for screening for analyte in a sample using the devices and apparatus are also provided. Samples suspected of containing analyte are added to a disposable self-contained sample tray containing one or more sample wells, mixed with a reagent, drawn into one or more spaced-apart capillary tubes held within a disposable cartridge connected to an analytical apparatus, reacted with a binding member on the surface of the capillary tube, washed to stop the reaction, and dried by the apparatus. The capillary tube is then exposed to a signal generation device to create a fluorescence signal that is detected using a signal detector. The apparatus determines the presence of the analyte and optionally determines the amount of analyte present in the sample, and presents the results to the operator.

CROSS-REFERENCE

This is a continuation-in-part of co-pending U.S. patent applicationSer. No. 08/254,302 filed Jun. 6, 1994, which application isincorporated herein by reference in its entirety now U.S. Pat. No.5,624,850.

BACKGROUND

1. Field of the Invention

This invention relates generally to immunoassays (particularlysolid-phase fluorescent immunoassays--SPFIAs), to devices for detectinganalytes by immunoassay using capillary tubes, to an apparatus for usewith such devices, and to manual, semi-automated, and automated, methodsfor such testing.

2. Background of the Invention

Many situations exist where qualitative, semi-quantitative, orquantitative detection of the presence of an analyte in a sample isdesired. Situations where analyte detection is desirable arise indiverse industries, including: 1) the health care industry, e.g., inclinical and diagnostic medicine (e.g., in vitro analysis); 2) the foodprocessing and chemical industries, e.g. in quality control for foodproduction; and 3) the environmental control industry, e.g. monitoringfor the presence of various pollutants in air, ground water, or soil.

Many assays using unique devices and protocols to detect the presence ofanalytes through chemical and physical means have been developed.Immunoassays make up one broad field of assays which find use in thedetection of analytes. In immunoassays, the occurrence of binding eventsbetween specific binding pair members is used as an indication of thepresence of analyte in the sample. Benefits of using immunoassays, ascompared to non-immunoassays, in analyte detection include highsensitivity, high specificity, reliability, and relatively short assaytimes.

The binding events that are utilized in immunoassays often occur at thesurface of a solid support with one binding member held at the surfaceof the solid support and the other binding member in the sample. Thetime required for a particular immunoassay to be completed will dependon the ability of the binding member in the sample to reach and bind tothe member on the support surface. The ability of the binding member inthe sample to bind with its pair on the support surface is dependent onmany factors; such factors include the concentration of the bindingmember on the support surface, and the surface to volume ratio of thesample/support combination. One method to decrease the time required foran immunoassay is to increase the concentration of a binding member on asupport surface. Another approach is to increase the ratio of thesurface area of the support relative to the volume of the sample to beassayed.

Common immunoassays include radio immunoassays (RIA), enzyme-linkedimmunosorbent assays (ELISA), and membrane based assays, such as commonhome pregnancy tests. These immunoassays have several disadvantages. Asignificant disadvantage of RIA is the requirement for the use ofhazardous radioactive isotopes. A disadvantage of ELISA is the numeroussteps of sample addition, incubation, washing, addition of colorreagent, addition of stop reagent, and reading required to perform theassay; such manipulations can be especially troublesome and a source ofsignificant error in the field. Also, enzyme reactions tend to betemperature sensitive, which require temperature control. Unlike RIA, inan which the label is directly detected, in an ELISA the enzyme label isnot directly detected. Instead, one must allow for a detectable productto be produced. Further, ELISA protocols may not be suited for allassays on all types of liquids, such as where the liquid comprising theanalyte of interest contains contaminants which interfere with one ormore individual steps in the assay, e.g. enzyme activity, detectabilityof enzyme product, and the like. Additionally, the safety concerns withRIA and the complexity of ELISA typically require that they be performedby relatively highly trained personnel and further require constantmonitoring by or interaction with the trained operator. A disadvantagewith membrane based assays is that they often provide poor quantitationand sensitivity. Many of these assays also require long incubationtimes, typically in the tens of minutes to hours, making analysis ofmultiple samples time consuming and expensive.

Nevertheless, ELISA is a commonly used format. In ELISA, binding eventsof interest are detected through the appearance of detectable productproduced by an enzyme acting on a substrate. The formation of thedetectable product can be amplified to the extent required by increasingthe concentration of the substrate and/or increasing the reaction time.On this basis, there is an opportunity to greatly increase the signalwhen only a few enzymes becoming bound.

Conventionally, ELISA has been conducted in microtiter plates consistingof wells. In an effort to improve performance, ELISA has beendemonstrated in capillary tubes. With ELISA immunoassays conducted incapillary tubes, rapid quantitative results are reported. At least onereported ELISA is described as sensitive and able to detect smallamounts of analyte. See e.g. Chandler et. al., "A new enzyme immunoassaysystem suitable for field use and its application in a snake venomdetection kit" Clinica Chimica Acta, 121:225-230 (1982). However, theaforementioned disadvantages inherent in ELISA still exist.

Additionally, while ELISA assays have been performed in capillary tubesin the laboratory, no successful products have been developed. A primaryreason for this is the difficulty of bringing several solutions into andout of the capillary tubes and the ability to effectively read theresult.

Therefore, there is a significant need for a fast, reliable, accurateimmunoassay that requires minimal interaction with the operator. Thereis also a need for an immunoassay that can screen several similar ordifferent samples sequentially or simultaneously for the same analyte orwhich can screen for different analytes in the same sample or in aplurality of aliquots of the same sample.

OBJECTS OF THE INVENTION

An object of this invention is to provide a simple, semi-automatedmethod for detecting and quantifying an analyte in a sample.

It is a further object of this invention to provide a solid-phasefluorescence immunoassay (SPFIA) that has fewer manipulations than acomparable enzyme-linked immunosorbent assay (ELISA) for detecting ananalyte in a sample.

A further object of this invention is to provide a rapid SPFIA thatindependently and semi-quantitatively or quantitatively assays for aplurality of analytes from a plurality of samples or aliquots from thesame sample.

A further object of this invention is to provide a reliable SPFIA thatrequires minimal manipulation of equipment by an operator performing theimmunoassay.

A still further object of this invention is to provide a SPFIA thatindependently and semi-quantitatively or quantitatively assays for aplurality of analytes from a plurality of samples in less than about 5minutes.

A still further object of this invention is to provide a uniquecapillary tube suitable to be used to achieve the aforementioned objectsof this invention.

A still further object of this invention is to provide a method forpreparing a capillary tube to be used to achieve the aforementionedobjects of this invention.

A still further object of this invention is to provide auniquely-designed cartridge for carrying at least one capillary tube(and preferably more than one) that can be used to achieve theaforementioned objects of this invention.

A still further object of this invention is to provide a tray having areservoir and a plurality of wells for holding a plurality of aliquotsof a sample, which tray can be used in conjunction with thecartridge-held capillary tubes to assist in achieving the aforementionedobjects.

A still further object of this invention is to provide an apparatus tobe used in conjunction with the cartridge-held capillary tubes andsample tray to perform the SPFIA of this invention and to further assistin achieving the aforementioned objects of this invention.

Other objects will be apparent to one of ordinary skill in the art uponreading the follow specification and claims.

SUMMARY

The present invention provides devices for screening for one or moreanalytes in a sample comprising capillary tubes, a cartridge, and asample tray which can combine to form a portable and disposable testingkit, and an apparatus and process for screening for one or moreanalytes, and a method for preparing capillary tubes for use with themethod for screening for one or more analytes, that are directed to thedisadvantages of the prior art and address heretofore unmet needspreviously discussed.

One aspect of the present invention is a cartridge for securely holdinga plurality of spaced-apart capillary tubes. The cartridge comprises aframe for holding the tubes in a spaced-apart manner, wherein the framehas a pathway in which each capillary tube can be aligned; and at leastone region in the frame to expose at least a portion of each capillarytube so that an electromagnetic signal can contact a portion of eachtube.

Another aspect of the present invention is a tray for holding multipleportions of a sample. The tray comprises a reservoir sufficient to holda quantity of fluid and a shelf extending substantially perpendicularlyoutward from a sidewall of the reservoir, the shelf having a pluralityof spaced-apart wells therein.

Another aspect of the present invention is a process for screening foran analyte in a sample. The process comprises importing a fluid mixtureinto a capillary tube coated on at least a portion of its interiorsurface with a substrate, wherein the fluid mixture comprises a samplesuspected of containing the analyte and a reagent comprising afluorescently-labeled conjugate that is (a) capable of binding to theanalyte or to the analyte and the substrate and (b) capable offluorescing when irradiated with an appropriate electromagnetic signal;maintaining the fluid mixture in the capillary tube for a timesufficient for binding to take place between the substrate and thefluorescently-labeled conjugate; removing excess fluid mixture from thecapillary tube; externally irradiating the coated portion of thecapillary tube with an electromagnetic signal sufficient to causefluorescence of bound fluorescently labeled conjugate; and detecting theresulting fluorescence to screen for the analyte.

Still another aspect of the present invention is an apparatus forscreening for at least one analyte in a sample. The apparatus comprisesa reservoir for a fluid; a conduit to transport the fluid to a port; theport being positioned to draw the sample thereto and to pump fluidtherethrough; a means to draw at least a portion of the sample to theport; a means to pump the fluid through the port; a first section havingconnecting means for a cartridge holding at least one capillary tube sothat one end of the capillary tube is in fluid communication with theport; a second section having means to hold a tray having at least onewell to communicate with the other end of the capillary tube, the secondsection also having a means to create a changing magnetic field so thata magnetizable metallic object held within the well of the tray is movedsufficiently to agitate a sample when placed in the well; a means tohold the cartridge and capillary tube to permit the capillary tube to beexposed to a signal generation means; the signal generation means; and asignal detection means positioned to detect a signal emitted from thecapillary tube as a result of exposure to the signal from the signalgeneration means.

Another aspect of the present invention is a combination of a cartridgeholding at least one capillary tube. The combination comprises acapillary tube coated on at least a portion of its interior surface witha substrate that is capable of binding to a fluorescently-labeledconjugate and a frame comprising a means for positioning the capillarytube in an exposure region of the frame, wherein the exposure regionpermits exposure of at least a portion of the coated capillary tube toan external electromagnetic signal that is capable of causing boundfluorescently-labeled conjugate to fluoresce.

Another aspect of the present invention is a capillary tube comprising asubstrate on at least a portion of its interior surface, which substrateis capable of being bound to a fluorescently-labeled conjugate.

Still another aspect of the present invention is a process for preparinga glass capillary tube for use in a fluorescent immunoassay. The processcomprises coating at least a portion of the internal surface of thecapillary tube with a substrate that is capable of binding to afluorescently-labeled conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a preferred embodiment of the inventionshowing cooperation between a cartridge containing capillary tubes and asample tray and cartridge holder combination when in use.

FIG. 2 is a perspective view of a preferred embodiment of the inventionshowing cooperation between the sample tray and cartridge duringstorage.

FIG. 3a is a perspective view of the obverse side of the fully assembledcartridge of a preferred embodiment of the invention showing elements ofthe design and the capillary tubes held therein.

FIG. 3b is a perspective view of the reverse side of the fully assembledcartridge of a preferred embodiment of the invention showing elements ofthe design and the capillary tubes held therein.

FIG. 3c is a perspective view of the reverse side of the fully assembledcartridge of a preferred embodiment of the invention showing elements ofthe design and the capillary tubes held therein, and particularlyshowing the distal portions of passageways for holding the capillarytubes.

FIG. 3d is a top view of the obverse side of the fully assembledcartridge of a preferred embodiment of the invention showing elements ofthe design and the capillary tubes held therein.

FIG. 3e is a top view of the reverse side of the fully assembledcartridge of a preferred embodiment of the invention showing elements ofthe design and the capillary tubes held therein.

FIG. 3f is view of the top or proximal end of the fully assembledcartridge of a preferred embodiment of the invention showing elements ofthe design and particularly details of the design of the top of the capthat forms a component of the cartridge.

FIG. 3g is a view of the bottom or distal end of the fully assembledcartridge of a preferred embodiment of the invention showing elements ofthe design and the capillary tubes held therein.

FIG. 3h is a side view of the fully assembled cartridge of a preferredembodiment of the invention showing elements of the design and thecapillary tubes held therein.

FIG. 4 is an exploded view of the various components of a preferredembodiment of the invention showing cooperation among elements.

FIG. 5 is a perspective view showing a distal orientation of the reverseside of a frame that forms a component of a preferred embodiment of thecartridge of the invention.

FIG. 6 is a perspective view showing a proximal orientation of thereverse side of the frame that forms a component of a preferredembodiment of the cartridge of the invention.

FIG. 7 is an exploded view of the top side of the holder that forms acomponent of a preferred embodiment of the cartridge of the inventionshowing cooperation between a receptacle and a cap.

FIG. 8 is an exploded view of the underside of the holder that forms acomponent of a preferred embodiment of the cartridge of the inventionshowing cooperation between the receptacle and the cap.

FIG. 9 is a perspective view of a preferred embodiment of the sampletray of the invention showing sample wells, a reservoir, and a cartridgestorage compartment.

FIG. 10 is a perspective view of a preferred embodiment of the sampletray of the invention showing the various components of the cartridgestorage compartment.

FIG. 11 is a perspective view of an alternative embodiment of the sampletray.

FIG. 12 is a perspective view of a preferred embodiment of an apparatusfor use with preferred embodiments of the devices for determining thepresence of an analyte, particularly showing the relative position ofthe sample tray and cartridge.

FIG. 13 is a transparent view of a preferred embodiment depicted in FIG.12, showing critical internal components.

FIG. 14a is a block diagram of a preferred embodiment of the apparatusshowing cooperation among the various electronic parts.

FIG. 14b is a block diagram, corresponding to FIG. 14a, of a preferredembodiment of the apparatus showing component designations known tothose of ordinary skill in the art.

FIG. 15 is a simplified diagram of the solid-phase fluorescenceimmunoassay (SPFIA) principle of a preferred embodiment of the inventionemploying a coating of a capture binding member comprising an antigen ina competitive fluorescence immunoassay.

FIG. 16a is a representative plot of concentration in parts per billionto normalized fluorescence for calibration standards comprising knownamounts of Penicillin G.

FIG. 16b is a representative plot of concentration in parts per billionto normalized fluorescence for calibration standards comprising knownamounts of Amoxicillin.

FIG. 16c is a representative plot of concentration in parts per billionto normalized fluorescence for calibration standards comprising knownamounts of Ampicillin.

FIG. 16d is a representative plot of concentration in parts per billionto normalized fluorescence for calibration standards comprising knownamounts of Cloxacillin.

FIG. 17a is a representative plot of concentration in parts per billionto normalized fluorescence for calibration standards comprising knownamounts of Cephapirin.

FIG. 17b is a representative plot of concentration in parts per billionto normalized fluorescence for calibration standards comprising knownamounts of Ceftiofur.

DETAILED DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENTS

Definitions

The following definitions are provided to help interpret the disclosureand claims of this application.

Amount: The term amount as used herein refers to the concentration orquantity of analyte in a sample either relatively or absolutely.

Antibody: The term antibody as used herein refers to a protein thatrecognizes a particular epitope on its homologous antigen. The antibodycan be a capture binding member of a substrate comprising the surface ofa capillary tube or it can be a moiety of a fluorescently-boundconjugate. An antibody can be a polyclonal antibody, a monoclonalantibody or a genetically engineered molecule capable of binding thecorresponding member of a specific binding pair.

Antigen: The term antigen as used herein refers to any substance thatbinds specifically to an antibody, as defined herein. An antigen can bea capture binding member of a substrate comprising the surface of acapillary tube, or it can be a moiety of a fluorescently-boundconjugate, or it can comprise an analyte.

Analyte: The term analyte as used herein, refers to any substance orchemical constituent of a sample that is being analyzed. Analytesdetectable by an immunoassay can be any analyte capable of beingrecognized and bound by a specific binding pair member. An analyte canbe one of the binding members of a homologous antibody-antigen bindingpair, that is, the analyte can comprise an antibody or an antigen in animmunoassay.

Capillary tube: A capillary tube as used herein comprises a high surfaceto volume ratio. A capillary tube can have any suitable shape whereinthe longitudinal cross section of the internal walls can be defined by acylinder, oval, square, rectangle, or any suitable polygon anddimensions that are appropriate for use in this invention, as describedhereinafter.

Conjugate: The term conjugate as used herein refers to a compound thatcomprises two substances, wherein one of the substances is coupled tothe other. For example, a first conjugate can be coupled to a thirdsubstance to make a second conjugate comprising a first conjugate and athird substance, or a first conjugate can be coupled to a secondconjugate to make a third conjugate comprising two conjugates. Couplingcan be covalent or non-covalent.

Fluorescent label: The term fluorescent label as used herein refers to asubstance which, when stimulated by an appropriate electromagneticsignal or radiation, will absorb the radiation and emit a signal thatpersists only so long as the stimulating radiation is continued, i.e. itfluoresces.

Fluorometer: The term fluorometer as used herein, refers to aninstrument for measuring fluorescence. Generally it comprises a signalgeneration means (i.e., a source of electromagnetic radiation of asuitable wavelength to cause a fluorescent label to fluoresce), a signaldetection means comprising a fluorescence detector, and an appropriatefilter or filters.

Immunoassay: The term immunoassay as used herein, refers to a techniquethat makes use of the specific binding between an antigen and itshomologous antibody, either polyclonal or monoclonal, in order toanalyze for an analyte in a sample. If the immunoassay comprises use ofa fluorescent label that can be detected when excited by an appropriateelectromagnetic signal, the immunoassay is a fluorescent immunoassay orFIA.

Reagent: The term reagent as used herein refers to a substance thatparticipates in a chemical reaction or physical interaction. A reagentcan comprise an active component, that is, a component that directlyparticipates in a chemical reaction (e.g. covalent binding) or physicalinteraction (e.g. non-covalent binding), such as a fluorescently-labeledconjugate, and other materials or compounds directly or indirectlyinvolved in the chemical reaction or physical interaction. It caninclude a component inert to the chemical reaction or physicalinteraction, such as catalysts, stabilizers, buffers, and the like.

Specific binding: As used herein specific binding refers to the chemicalrecognition between two substances that results in the coupling of thosesubstances. Such coupling can include, but is not limited to, covalentor non-covalent interaction. For example, the specific binding betweenan antigen and its corresponding antibody is of greater affinity thanthe non-specific binding of either specific binding pair member and anon-corresponding substance.

Specific binding pair: As used herein specific binding pair refers totwo substances that specifically bind to each other.

Specific binding pair member: Specific binding pair member, as usedherein, refers to one member of a specific binding pair. For example, aconjugate, a hapten, an antigen or an antibody can be a specific bindingpair member.

Substrate: The term substrate as used herein refers to a material towhich another material binds or can be attached. Such a material isgenerally a surface of a material (e.g., the interior surface of acapillary tube), a solid material on a surface, or a first solidmaterial on a second solid material. Generally a substrate can comprisea capture binding member of a binding member pair that can bind to asecond member of the binding pair. For example, a substrate can be anantibody conjugated with a substance that binds to the interior surfaceof a capillary wall, wherein the antibody would be considered a capturebinding member of a binding pair. The corresponding antigen conjugatedwith a fluorescent label would be the second binding member.

Introduction

In one broad aspect, this invention can be considered to be animmunoassay that employs the interior surface of a capillary tube as asolid phase substrate that can bind a fluorescently-labeled conjugateand detect an analyte in a sample. Another aspect of this inventionincludes a capillary tube coated on at least a portion of its interiorsurface with a substrate that is capable of binding with afluorescently-labeled conjugate. This unique capillary tube provides, atleast in part, the basis for other aspects of the invention, such as acombination of the capillary tube with a cartridge and a process forscreening for an analyte in a sample using the capillary tube. Anotheraspect of the invention is a process for preparing the capillary tube ofthe invention. Still other aspects of the invention include a uniquelydesigned sample tray for use with the cartridge-capillary tubecombination and an apparatus for performing the screening process ofthis invention. These and other aspects of the invention will bediscussed in greater detail hereinafter.

Types of Analytes to be Detected

The immunoassay of this invention is useful for screening for analytesin numerous industries including, but not limited to, health care, foodprocessing, chemical, environmental control, and the like. Thus, thetypes of samples include various fluids suspected of containing a targetanalyte such as blood, plasma, urine, saliva, and the like; foodproducts such as milk, wine, beer, and the like; chemical streams, wastestreams from chemical plants, rivers, and the like. In some situations,the sample may need to be pre-treated prior to the immunoassay. Wherethe sample is initially complex, solid, or viscous, it may need to beextracted, dissolved or diluted in order to obtain a sample having theappropriate characteristics for use in the immunoassay. Further, thesample should be one in which binding complexes formed in the subjectimmunoassay are stable. Binding complexes of the subject immunoassaywill generally be stable at pH values ranging from about 5 to about 9.The pH value of the sample may be adjusted, if necessary, to be about 7by diluting with an appropriate buffer.

A wide range of analytes can be detected using the subject method.Detectable analytes can be any analyte capable of being recognized andbound by a specific binding pair member. The analyte can be an antigen,and antigen receptor (e.g. an antibody), or hapten. Analytes of interestinclude naturally occurring and synthetic small organic compounds,proteins, saccharides, nucleic acids and the like. Illustrative analytesinclude compounds used in the raising of domestic animals (e.g.antibiotics and food supplements), food additives, naturally occurringcontaminants, dyes, microorganisms and their toxins, and the like. Otheranalytes include physiologically active compounds, pathogenic markersfound in physiological fluids, toxins, surface membrane proteins,cytokines, antibodies, human lymphocyte antigen proteins, hormones,natural and synthetic drugs, proteins of bacteria, fungi and viruses,and the like. Other analytes include compounds found in the environment,such as pesticides, herbicides, organic components of waste discharges,and the like.

Examples of specific analytes detectable in the immunoassay of thisinvention include compounds from the sulfa family drugs such assulfamethazine, sulfadimethozine, sulfathiazole, sulfaquinoline andothers; tetracycline, gentamicin, chloramphenicol, aflatoxin, digoxin,and salmonella. Still other analytes include, hydrocarbons, such asbenzene, toluene, ethyl-benzene, and xylenes.

The immunoassay is particularly valuable for detecting analytes, such asantibiotics in milk samples, and is presently preferred to be used forthese compounds. Antibiotics are administered to cows for the preventionand treatment of infections, such as mastitis, and to enhance animalgrowth and milk production. Antibiotics are also abused throughoff-label, illegal administration in an attempt to quickly bring a sickanimal back into the producing herd.

For the purpose of maintaining a safe and healthy food supply, it isimportant to identify, monitor and minimize the existence of antibioticresidues in milk and milk products. Since milk and milk products arewidely consumed, antibiotic residues should be avoided for severalreasons: (i) Some antibiotic residues can cause allergic reactions insensitive consumers (approximately 5 to 10% of the population ishypersensitive to penicillin and other antibiotics), (ii) Smallconcentrations of antibiotic residues can aid in the selection ofresistant strains of pathogens that are harmful to humans, and (iii)Antibiotic residues can interfere with starter cultures used in theproduction of processed milk products such as cheese and yogurt.Consequently, the United States Food and Drug Administration (US FDA)has established safe/tolerance levels for antibiotic residues in milkand milk products, shown in Table I.

The antibiotics that are most commonly administered to lactating cows,and consequently those antibiotics that are usually found ascontaminants in milk and milproducts are from the β-lactam group(penicillin G, ampicillin, cloxacillin, cephapirin, ceftiofur, andamoxicillin). The structures for these compounds are available byconsulting the "Merck Index"--Eleventh Edition.

                  TABLE I                                                         ______________________________________                                        U.S.F.D.A. Safe/tolerance levels for β-lactam drugs in milk                    Drug      Safe/Tolerance Level (ppb).sup.a                              ______________________________________                                        Penicillin G                                                                               5                                                                  Amoxicillin 10                                                                Ampicillin 10                                                                 Cloxacillin 10                                                                Cephapirin 20                                                                 Ceftiofur 50                                                                ______________________________________                                         .sup.a ppb = parts per billion, 1 ppb is equal to 1 ng/mL.               

The Immunoassays

Depending on the type of analyte to be detected, different immunoassayformats can be employed. A particular immunoassay format can be modifieddepending on the nature of the analyte, the nature of the sample, andthe like. In general, immunoassays useful in the subject method arebased on the formation of complexes between specific binding pairmembers. Common to each immunoassay used in the subject method will be asubstrate comprising a first binding pair member. The immunoassay alsoemploys a second binding pair member comprising a fluorescently-labeledconjugate that can couple to an analyte or to both the first bindingpair member and an analyte. Throughout this specification, the term"capture" is used with binding member or specific binding pair member.It refers to a specific binding pair member comprising a substrate onthe interior surface of a capillary tube, although it is not limited tosuch use if indicated otherwise.

Both sandwich and competitive immunoassay formats can be employed in thesubject method. The particular immunoassay format employed will dependon the particular analyte characteristics, the sample characteristics,the available reagents, and the like.

In a sandwich immunoassay, a fluorescently-labeled conjugate is employedthat is a specific binding member, wherein the fluorescently-labeledconjugate binds to the analyte at a site other than the site to whichthe other binding member, which is on the interior surface of thecapillary tube. The fluorescently-labeled conjugate is mixed with asample and the resulting mixture is drawn up into the capillary tube.The analyte will bind to the fluorescently-labeled conjugate and to theother binding member moiety of the substrate, so that the amount offluorescent label bound to the capillary tube wall will be directlyproportional to the amount of analyte present.

In one type of a competitive immunoassay, a fluorescently-labeledconjugate binding member binds directly to an analyte or to thesubstrate, via an analog of the analyte present on the substrate. Thus,the analyte and the substrate "compete" to bind with thefluorescently-labeled conjugate binding member. In this format, both thesubstrate and the analyte have a binding region (also referred to as anepitopic region) that bind to the fluorescently-labeled conjugatebinding member. The moiety of the substrate can be a ligand, anantibody, or binding fragment thereof, typically analogous to anepitopic region of the analyte. Here the amount of fluorescent labelbound to the interior wall of the capillary tube is inverselyproportional to the amount of analyte present.

In another type of a competitive immunoassay, a fluorescently-labeledconjugate competes with the analyte for binding sites to the capturebinding member of the substrate. In this format, the capture bindingmember can comprise an antibody with both the analyte and thefluorescently-labeled conjugate comprising homologous antigen bindingmembers. In the absence of analyte, the fluorescently-labeled conjugatewill not have competition for binding sites on the capture bindingmember of the substrate. Thus, the amount of fluorescent label bound tothe interior wall of the capillary tube is inversely proportional to theamount of analyte present.

With this in mind, it can be seen that an aspect of this invention is aprocess for screening for an analyte in a sample, which processcomprises importing a fluid mixture into a capillary tube having asubstrate comprising a capture binding member wherein the fluid mixturecomprises a sample suspected of containing the analyte and a reagentcomprising a fluorescently-labeled conjugate that is (a) capable ofbinding to the analyte or to the analyte when bound by the capturebinding member and to the substrate; (b) capable of fluorescing whenirradiated with an appropriate electromagnetic signal. The processfurther comprises maintaining the fluid mixture in the capillary tubefor a time sufficient for binding to take place between the substrateand the fluorescently-labeled conjugate; removing excess fluid mixturefrom the capillary tube; externally irradiating the coated portion ofthe capillary tube with an electromagnetic signal sufficient to causefluorescence of bound fluorescently-labeled conjugate; and detecting theresulting fluorescence to screen for the analyte.

In a competitive immunoassay, a particularly convenient protocol iswhere fluorescently-labeled conjugate is first mixed with a liquidsample suspected of containing the analyte to provide a substantiallyhomogeneous mixture and then the sample is taken into the capillarytube. The fluorescently-labeled conjugate can be a solid, or preferably,a buffered solution, and as appropriate, can serve to dilute the sampleand provide the appropriate pH.

Once the sample suspected of containing the analyte has been mixed withthe appropriate fluorescently-labeled conjugate, the resulting mixtureis introduced into the interior of a capillary tube that has beeninteriorly coated with an appropriate capture binding pair member moietyof a substrate appropriate for the analyte being assayed. Preferably, asample is introduced by capillary force, although an external force suchas suction or positive pressure can also be used. In the examplereferred to above, an antibiotic in milk, an antigen analogous to anantibiotic of interest, is coated on the interior surface of thecapillary tube. The entire interior surface or a part thereof can becoated. However, enough of the surface must be coated so that a bindingreaction can take place between the fluorescently-labeled conjugate andthe capture binding member moiety of the substrate such that thecapillary tube can be read by irradiating it and detecting the resultingfluorescence. Generally, sample volumes introduced into the capillarytube will range from about 2 to about 20 μl, usually about 5 to about 15μl, more usually about 5 to about 10 μl.

After the sample portion has been introduced into the capillary tube,the sample is incubated for a sufficient time period for binding tooccur, that is to form complexes between members of specific bindingpairs, e.g. a fluorescently-labeled conjugate binding member and thesubstrate comprising the bound antigen. The incubation step willtypically occur at room temperature, although temperatures in the rangeof about 10° C. to about 50° C. can be employed. Incubation times willtypically range from about 0.5 to about 5 minutes, usually about 0.5 toabout 3 minutes, and more usually about 2 minutes. Frequently, the timenecessary for introducing a wash solution into the capillary tube willsuffice for the incubation.

For the most part, the subject methods will depend solely on thecapillary tube and the fluorescently-labeled conjugate for carrying outthe immunoassay. However, in some situations more complex protocols canbe employed. For example, instead of having the conjugate binding memberlabeled directly, one can indirectly label the binding member. Where thebinding member is an antibody, one can use a fluorescently-labeledanti-antibody, so as to have a universal fluorescent reagent. One canhave a situation where one adds both a fluorescently-labeled conjugateand its reciprocal binding member, where the conjugate competes with theanalyte for the reciprocal binding member. The capillary tube cancomprise a capture binding member that captures the reciprocal bindingmember. For example, the reciprocal binding member can be an antibodyand the capillary tube can be coated with Protein A or G, so as tocapture all antibodies.

After an incubation step, any fluorescently-labeled conjugate free inthe medium is preferably removed from the capillary tube. Removal ofunbound fluorescently-labeled conjugate is conveniently accomplishedthrough introduction of a washing fluid that displaces unboundfluorescently-labeled conjugate from the capillary tube. A variety ofwash fluids can find use for the washing step. The pH of the wash fluidwill be a pH in which the binding pair complexes are stable. Typically,the pH will range from 5 to 9, usually 6 to 8, and more usually about is7. Depending on the nature of the fluorescent label of the conjugate,wash solutions which enhance the fluorescence of the conjugate label canbe employed. For example, the fluorescence of a particular fluorescentlabel can be enhanced in slightly alkaline or basic solution. In such acase, a buffer having a pH above 7, but usually less than 9, can beemployed. Exemplary wash fluids comprise water, buffers, such asphosphate, phosphate buffered saline (PBS), saline solutions, carbonatebuffers, and the like. The wash fluid can be introduced into thecapillary tube using any convenient means. Usually the wash fluid willbe introduced into the capillary tube using the same means as the meansused for introduction of the sample. The wash solution can be taken up anumber of times, usually not more than about 6, more usually not morethan about 2, or the wash solution can be forced through the capillarytube using a syringe, pump or other device.

After the washing step where the unbound labeled conjugate is washedfrom the capillary tube, the presence of fluorescently-labeled conjugateremaining bound to the capture binding member on the substrate on thecapillary tube surface is detected in a detection step. The detectionstep can be conducted immediately after the wash step, or can be delayedfor a period of time, if necessary. While the detection step can beconducted with wash fluid in the capillary tube, preferably thecapillary tube can be dried prior to the detection step to minimize thepossibility of interference in the ensuing detection step. The dryingmay be done by any appropriate means such as centrifuging, air drying,vacuum drying and the like. Preferred techniques are discussedhereinafter. If the detection step is to be delayed, the capillary tubecan be stored for a reasonable period of time under ambient or reducedtemperature conditions.

Many different fluorescent labels can be employed in the subjectimmunoassays. Suitable fluorescent labels should be capable ofconjugation with antigens, haptens or antibodies in order to be used inthe fluorescently-labeled conjugate. Selection of the fluorescent labelis based on synthetic convenience, emission maximum, quantum efficiency,stability under the assay conditions, and the like, but the fluorescentlabel is not critical to the invention, so long as there is a minimumquantum yield to provide the desired sensitivity. A large number ofcommercially available fluorescent labels can be employed. Illustrativefluorescent labels include fluorescein isothiocyanate (FITC), rhodamine,Texas Red, phycoerythrin, Cy-5® and allophycoerythrin, and particularly,fluorescent labels that fluoresce above about 550 nm, more particularly,fluorescent labels that fluoresce above 600 nm, and efficiently absorblight having absorption above 500 nm; more particularly, 650 nm, such asCy-5®. The fluorescent labels can be conjugated to form thefluorescently-labeled conjugate using any convenient method. See e.g.Harlow & Lane, Antibodies (1988) pp 353-358.

When fluorescently-labeled conjugates are used, detection isaccomplished by first irradiating a region of the capillary tubecomprising the detection region, followed by measuring the resultantemitted fluorescent signal. Any convenient irradiation means can beemployed for providing the appropriate wavelength. Exemplary irradiationmeans include lasers, light emitting diodes, tungsten lamps and thelike. The wavelength of light used in the stimulation means will dependon the particular fluorescent label. Generally, the irradiation lightwavelengths will range from 300 to 900 nm, usually from about 350 to 800nm, and more usually from about 450 to 800 nm. For example, where Cy-5®is the fluorescent label, the wavelength of the irradiation light willrange from 630 to 650 nm. The fluorescence from thefluorescently-labeled conjugates present in the capillary tube will bemeasured. Measuring the emitted signal is accomplished by detecting thephotons emitted in the detection region. Means for measuringfluorescence are commercially available and any convenient fluorescencedetector can be used. Various photodiodes, photomultipliers, and thelike, can be employed, and in some instances a visual detection willsuffice, if a fluorescently-labeled conjugate is used that fluoresces inthe visible spectrum.

Depending upon whether a competitive or sandwich immunoassay isemployed, and the reagents employed, the fluorescence intensity will bedirectly or inversely proportional to the amount of analyte in thesample. Where one is interested in a qualitative result or asemi-quantitative result, such as determining whether the amount ofanalyte is above a predetermined threshold, versus determining theconcentration of analyte, the amount of fluorescently-labeled conjugateis selected to provide a clear signal as compared to the absence ofanalyte or analyte below the predetermined value. Thus, one can use anamount of conjugate which will be substantially absent in the detectionregion in the absence of analyte and provide an intense signal at thelowest concentration that one would anticipate to be encountered ofanalyte in the sample or vice versa.

For quantitation, the resultant electrical signal can be accuratelymeasured using appropriate hardware and software. The area from whichthe fluorescence is measured is controlled to provide for consistentvalues. Controls can be employed, where the signal to concentration ofthe analyte is determined, so that the signal can be directly related tothe concentration of analyte in the immunoassay sample. In this manner,both the presence and the amount of analyte in the sample can bedetermined.

As discussed hereinafter, generally only a small amount of the sample(less than about 1 ml) is mixed with the conjugate. As discussed ingreater detail hereinafter, this is done preferably in a small well of asample tray using a means of agitation. It may be important that themixing and incubation step and the subsequent incubation step in themethod of this invention be carried out for the same defined period oftime to minimize variations from test to test in a series. Thus, it maybe preferred to automate the steps of the method to eliminate operatorerror to the greatest extent possible. This will be discussed in greaterdetail in the following discussion of preferred devices and apparatusdepicted in the figures.

Thus, from the preceding discussion, it can be seen that solid-phasefluorescence immunoassay (SPFIA) takes advantage of the specificity of abinding pair affinity, e.g., antibody-antigen recognition, andincorporates a suitable fluorescent label for detection. FIG. 15diagrams the principle of the SPFIA. A sample (e.g., milk) is mixed witha known amount of a fluorescently-labeled antibody which is specific toan analyte (e.g., a β-lactam antibiotic). An essentially instantaneousbinding reaction will occur between the antibody and the analyte in thesample. A solid support, e.g., the inside wall of a glass capillary tubeas a substrate comprising an antigen analogous to the analyte attachedthereto will then be exposed to the sample. When the sample is removedfrom the capillary tube and the capillary tube is examined with afluorometer, and any fluorescence is detected. In the milk example shownin FIG. 15, a substrate incubated with milk samples with highconcentrations of an antibiotic will have fewer freefluorescently-labeled antibody molecules to react with the antigensubstrate, thus yielding lower fluorescence signals. Conversely, milksamples with low concentrations of antibiotic will have more freeantibody molecules, thus more fluorescent conjugates will bind to thesubstrate and will provide higher fluorescence signals. Mathematically,the measured fluorescence signal is inversely proportional to theconcentration of analyte in the sample for these competitive assays.Thus, in FIG. 15 the analyte is the antibiotic and the capture bindingmember moiety of the substrate on the interior capillary tube surface isan antigen (e.g., an analog of the analyte) that is bound by thefluorescently-labeled antibody conjugate.

The Capillary Tube And Its Preparation

An important aspect of this invention is the unique capillary tubecoated on at least a portion of its interior surface with a substratethat is capable of binding with a fluorescently-labeled conjugate. Awide variety of capillary tubes designed for diverse uses are known inthe art and are suitable for use in the subject invention. Generally,capillary tubes used in the subject method are made of a substance thatallows irradiation light and fluorescence emissions to be transmittedacross the capillary tube wall. Thus, when an electromagnetic signalfrom an external source contacts the capillary tube wall, the signalmust go through the wall. The resulting fluorescence must then betransmitted out through the wall. Materials from which suitablecapillary tubes can be formed include glasses, such as soft glass,silicate glass and fused silica. Other materials include plastics, suchas polystyrene, polyethylene, polypropylene, polyvinyl chloride (PVC),and the like. Other materials may be apparent to one of skill in the artupon reading this disclosure. A presently preferred embodiment is aborosilicate glass capillary tube, such as the one available fromDrummond Scientific, Broomall, Pa.

Capillary tubes suitable for use in the subject invention can have awide variety of dimensions, as long as liquid media are effectivelydrawn up by suction or capillary force. The capillary tubes can havecross-sections which are circular, square, rectangular, oval, and thelike. Typically, the capillary tubes will have circular cross-sections.The inner diameters of suitable capillary tubes can range from about 0.1micrometers (μm) to about 1 millimeter (mm), usually about 0.3 μm toabout 1.0 mm and more usually about 0.50 μm to about 1 mm, preferablyabout 0.65 mm for a milk immunoassay. The outer diameter of a suitablecapillary tube can be about 1.0 mm to about 1.5 mm. The length ofsuitable capillary tubes will typically be about 10 mm to about 150 mm.Usually the length will be at least about 15 mm, more usually at leastabout 25 mm, up to about 250 mm for ease of handling. While thecapillary tubes of this invention may be interconnected by a planarsheet, generally they are free standing, individual capillary tubes.

For glass capillary tubes, to enhance binding of a substrate to theglass, the surface can be coated with a material to enhance a bindingcapability on the interior surface. At least one region of the interiorsurface of the capillary tube, a detection region, will be coated with asuitable substrate that will include a member of a binding pair. Themember of a binding pair coated on the surface, at the detection region,binds directly or indirectly through an intermediate binding agent to afluorescently-labeled conjugate. Depending on the particular method usedto coat the capillary tube, the region can encompass the entire interiorsurface of the capillary tube or can be limited to a portion of theinterior surface of the capillary tube. The region of the interiorsurface comprising a capture binding member coated thereon willtypically range from about 10 to 100% of the interior surface, and willusually range from about 30 to about 100% of the interior surface.Preferably more than about 80% of the interior surface is coated.Conveniently, the entire capillary tube, or one end thereof, can beimmersed in the coating media to be coated. When immersed at one end,the coating medium can be brought up into the capillary tube by anysuitable means, e.g. by capillary force, conveniently to a predeterminedheight, which can be indicated by a scoring or other designation on thecapillary tube. Alternatively, the liquid can be pumped or sucked into aportion of or the whole length of the capillary tube. This means thatall or a portion of the external surface can be coated with the capturebinding member as well.

Depending on the particular immunoassay format used in the subjectmethod, a variety of agents can serve as the binding members. Ingeneral, a binding member of a pair should complex or bind to itscomplementary binding pair member in the subject immunoassays withsufficient affinity to withstand wash procedures used in the subjectmethod. Typically, the affinity between the binding member and itscomplementary binding pair will be at least about 10⁶ L/mol, frequentlyat least about 10⁸ L/mol or higher. One member of a binding pair willbind or complex directly or indirectly to a fluorescently-labeledconjugate. For example, in a competitive immunoassay format, the capturebinding member (e.g., the analyte analog on the interior surface of thecapillary) binds directly to a fluorescently-labeled conjugate. In asandwich immunoassay format, the capture binding member will complex tothe fluorescently-labeled conjugate indirectly through the analyte.Illustrative binding members include receptors, such as antibodies andbinding fragments thereof, e.g. F(ab) and F(ab)₂ fragments), lectin,ligands, such as antigens, haptens or other reciprocal binding members;and conjugates comprising ligands and receptors, bonded to thefluorescent label.

Instead of coating a region of the interior with a capillary tube with asubstrate containing a single capture binding member, the interiorsurface of the capillary tube can be coated with two or more capturebinding members at the same or different sites. In this embodiment, eachdifferent capture binding member will be involved in the detection of adifferent analyte in the sample. When the binding members are at thesame site, the fluorescent label associated with each analyte can beindependently determined, e.g. different emission maximum wavelength, atleast about 10 nm different, and/or different delay time for emission.Thus, one can assay for a multiplicity of analytes simultaneously.

To coat the internal wall of capillary tubes for use in the subjectmethod, capillary tubes are contacted with a solution comprising thecapture binding member. For coating the internal capillary tube surfacewith the substrate solution, a variety of techniques can be employed,depending in part on the nature of the substrate and the nature of theinternal wall. With most proteins, particularly antibodies, albumins andglobulins, the proteins stick to the surface without covalent bonding,and are stable under the conditions of the immunoassay.

In preparing the subject capillary tubes, as indicated for proteinsabove, it can be sufficient to contact capillary tubes with untreatedsurfaces to a solution comprising the binding reagent. The bindingsolution is usually a buffered solution having from about 10⁻⁷ to 10⁻³ gprotein/ml. Typically, the protein binding member will be an antibody orfragment thereof for direct assays. For indirect assays, the proteinwill typically be an analog of the analyte. For the most part, theprotein binding member will be an antibody or fragment thereof. Methodsof stably coating glass and plastic surfaces are well known. See e.g.Harlow & Lane, Antibodies: A laboratory manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1988). In many instances, wherethe binding member is not a protein, it can be conjugated to a protein,leaving the binding sites available for binding to the complementarybinding member. For example, haptens can be conjugated to a protein thatwill not interfere with the immunoassay. In this way, an otherwisenon-binding analyte or a mimetic or analog thereof can be directly boundto the internal surface of the capillary tube without having tofunctionalize the surface so as to provide covalent binding of theotherwise non-binding substance.

In the case of a preferred capillary tube, i.e., glass, it is preferredto first coat the interior surface with an agent which enhances thebinding of the protein to the surface. Thus, where the binding memberdoes not provide for stable binding to the interior surface of thecapillary tube, the surface is activated or functionalized to providecovalent or non-covalent binding of the binding member to the capillarytube surface. The particular technique used in treating the capillarytube surface will depend on the composition of the capillary tube andthe binding member, e.g. the functional groups available on the bindingmember for reaction. With surfaces such as plastics, e.g. polystyreneand polyethylene, the surface can be functionalized to provide forreactive amino, carboxy, thio, sulfonyl, hydroxy or other functionalgroups, by acylation, nitration and reduction, oxidation with ozone,chlorosulfonation, and the like. The specific functional group providedon the capillary tube surface will depend on the binding member. If thebinding member does not naturally comprise a useful available functionalgroup, the binding member can be modified, so as to provide for afunctional group that will react with the activated surface, e.g. aminowith carboxy, thiol with activated olefin, hydroxy with an activatedhalogen, and the like. For non-covalent binding of the binding member tothe surface, a hydrophobic surface may be provided, that is, a surfacethat has a long chain alkyl or alkenyl group attached, e.g., through asilicon attaching group.

For treating the surfaces of glass capillary tubes, the surface can be"functionalized" by using a silicon-based compound having as one part ofthe compound a silicon moiety that reacts with the glass surface of thecapillary tube and the other part of the compound being carbon-basedthat provides a suitable functional group, e.g. alkyl, alkenyl, amino,carboxy, sulfonyl, thiol, activated olefin, such as maleimido, and thelike, that will bind with the binding member either covalently ornon-covalently (e.g., by van der Waals' forces).

Generally, the coating is done using a silicon-based material thatprovides a basis for covalently or non-covalently forming a suitablesubstrate on the interior surface. Suitable silicon-based materialsinclude silanes or siloxanes that bind through the silicon to theinterior glass surface and provide a surface extended into the cylinderof the capillary tube to which an appropriate substrate is bound. Thisis seen in FIG. 15. Examples of suitable siloxane materials includeaminoalkylsiloxanes and alkyl or alkenyltrialkoxysilanes. Conveniently,aminoalkylsiloxanes known in the art can be used, where the aminoalkylgroup is of from about 2 to 6 carbon atoms and the alkoxy groups are offrom about 1 to 6 carbon atoms. Preferably the silane based material isrepresented by the formula R-Si(OR₁)₃, wherein R is an alkyl or alkenylof about 12 to about 20 carbon atoms and R₁ is an alkyl of one to fourcarbon atoms. A particularly preferred silane-based material is acompound represented by the formula R-Si(OR₁ )₃, wherein R is a straightchain alkyl of 18 carbon atoms and R₁ is ethyl. Preferablyoctadecyltriethoxy silane is chosen as the silane coating material, thisis available through Pierce Chemical Company as AquaSil®.

After the binding member has been bound to the surface of the capillarytube, non-specific active sites or "hot spots" can remain on thecapillary tube surface. These active sites must be occupied or blockedprior to use of the capillary tube in the subject method. Otherwise,non-specific adsorption of the various assay reagents can occur.Non-specific adsorption should be avoided because it can result in non-specific binding of the label to the surface. Blocking of active sitescan be achieved by contacting the internal capillary tube surface with awide variety of blocking solutions. Coating the capillary tube surfacewith the blocking solution can be achieved using the methods describedabove for coating the surface with the binding member. The blockingsolution of choice will depend on the particular immunoassay beingconducted. See e.g., Harlow and Lane, Antibodies (1988) 497, supra.Illustrative blocking solutions include Blotto, Blotto/Tween, Tween, BSAand Horse Serum.

Generally, silane-based coating is carried out by incubating anappropriate capillary tube in a solution containing the silane basedmaterial dissolved in an appropriate solvent, such as water, for aperiod of time and at a temperature sufficient to allow binding of thesilane material to the internal surface of the glass capillary tube. Anappropriate temperature range is about 10° C. to about 50° C, withambient temperature being preferable. The binding will take place withinabout 10 minutes, but the capillary tubes can be incubated for an houror more. The capillary tube is then dried via a suitable means such asby purging with nitrogen or other suitable gas or by centrifugation. Anyresidual moisture can be removed by incubating the capillary tube in anoven at a suitable temperature, such as about 110° C. to about 140° C.,for about an hour and/or under a vacuum. After incubation, the capillarytube will be allowed to cool to room temperature before treating it withthe next coating. Once the glass is alkylated by the silane material itis susceptible to non-covalent binding with sticky proteins. A substratecomprising a capture binding member, i.e. a protein conjugated to acapture binding member of an antigen-antibody binding pair, can becoupled to the silanization substrate layer. Alternatively, a proteinconjugated to another protein substance that permits binding to a thirdsubstrate comprising the capture binding member can be applied. Whencoating a protein or conjugated protein to the silanization layer, alonger incubation time will usually be required, typically between about1 to about 24 hours for a similar period of time. After coating withappropriate substrate layers, a final coating with an innocuous proteinblocking solution is applied to cover "hot spots" previously discussedcomprising exposed alkyl groups. Alternatively, the blocking step canoccur after the attachment of the second layer, but before attachment ofa third layer in cases where a third layer is used. The coated andblocked capillary tubes are typically incubated in the blocking solutionfor at least 1 hour and as long as 24 hours. The capillary tube is thensubstantially dried. A wash solution, such as deionized water, is thenpassed through the capillary tube to remove any substrate material notbound to the surface of the capillary tube or to another substrate. Thecapillary tube is then substantially dried by any appropriate means. Itis advantageous to aid removal of residual water by incubating thecapillary tube in a vacuum oven at the highest temperature possible thatwould not denature any bound protein, e.g. about 37° C. for at least onehour. Once the capillary tubes have been prepared, they can be usedimmediately or conveniently stored for use at a later time. Capillarytubes can be stored for use at a later time at ambient or reducedtemperature conditions. Example 9, Example 10, and Example 11, describein detail, preparation of capillary tubes for use with a preferredembodiment of the invention.

Another aspect of this invention comprises a process for preparing aglass capillary tube for use in a fluorescent immunoassay, which processcomprises coating at least a portion of the internal surface of thecapillary tube with a substrate that is capable of binding to afluorescently-labeled conjugate. In a preferred aspect, the processcomprises coating at least a portion of the interior surface of thecapillary tube with a silane-based material and binding a proteinconjugate to the silane-based material, which conjugate is capable ofbinding to a fluorescently-labeled conjugate. Protein conjugates thatare particularly useful include bovine serum albumin (BSA) and humanserum albumin (HSA), the former being preferred.

A Cartridge For Use With Capillary Tubes

Capillary tubes prepared by the methods described above can be usedindividually or in combination for an immunoassay in accordance with themethod discussed above. The assay can be performed manually orautomatically, as discussed hereinafter. One end of a single capillarytube can be introduced into a sample and sample drawn up by anyconvenient means, e.g. capillary action or active pumping, to provide anappropriately sized sample in the capillary tube. Alternatively, aplurality of capillary tubes coated with appropriate binding members canbe used to permit a plurality of immunoassays on one sample or a singleimmunoassay on multiple samples to screen for one or multiple analytes.The capillary tubes can be held in a cartridge that permits sequentialor simultaneous use and/or that permits interaction with other devicesand/or apparatus to effectuate an immunoassay of this invention.

Thus another aspect of this invention is a combination of a cartridgeholding at least one capillary tube, which cartridge comprises acapillary tube coated on at least a portion of its interior surface witha substrate that is capable of binding to a fluorescently-labeledconjugate and a frame comprising a means to position the capillary tubein a region of the frame that permits exposure of at least a portion ofthe coated capillary tube to an external electromagnetic signal that iscapable of causing any bound fluorescently-labeled conjugate tofluoresce. The fluorescence is then detected with an appropriate signaldetection means.

Another related aspect of this invention is a cartridge for securelyholding a plurality of spaced-apart capillary tubes, wherein thecartridge has a plurality of passageways for aligning the capillarytubes therein and at least a portion of the cartridge permits exposureof the capillary tubes to an external electromagnetic signal that iscapable of causing a fluorescently-labeled conjugate on the internalsurface to fluoresce. The cartridge preferably has a holder with apassageway therethrough and a port for permitting fluid to be passedthrough each capillary tube. The cartridge can be manufactured bymethods known to those of ordinary skill in the art, and is preferablyinjection molded. The cartridge is particularly suitable for cooperatingwith a unique sample tray and with a semi-automated apparatus forfluorescence immunoassays, as will be discussed in more detailhereinafter.

A detailed description of a preferred embodiment of the cartridge isprovided with the description of the drawings and alternativeembodiments are provided thereafter.

A Sample Tray For Use In Immunoassays

Capillary tubes as prepared above can be used with commerciallyavailable containers suitable for independently housing the reagents andwaste of immunoassays, such as microtiter wells or plates.Alternatively, the capillary tubes can be used with a sample traydesigned to contain a reagent and means to mix fluid held within thewells.

Another aspect of this invention, therefore, is a tray for holdingmultiple portions of a sample, which tray comprises a reservoirsufficient to hold a quantity of fluid, and a plurality of spaced apartwells therein. Preferably, the sample tray also independently houses ametallic washer for mixing any fluid held within the wells, and has ameans to prevent the metallic washer from falling out of the well,preferably a retaining ridge with a diameter smaller than the externaldiameter of the metallic washer. The sample tray may be manufactured byany number of methods known to those of ordinary skill in the art and ispreferably injection molded.

A preferred aspect of this invention is the combination of the sampletray and cartridge to form a disposable immunoassay testing kit. In thisaspect, the sample tray has a section for securely holding the cartridgetherein for storage and transportation. Details of the sample tray andcartridge and sample tray combination are provided in the description ofthe figures.

An Apparatus For Performing the Process for Screening for Analytes

The methods of the present invention for screening for analytes can beconducted manually. The steps of washing and drying can also beconducted manually. The capillary tube can also be used in accordancewith the invention with a commercially available fluorometer.

However, it is preferred that the methods be conducted by an automatedor semi-automated apparatus. Since, incubation times are relativelyshort for the SPFIA in capillary tubes, a greater probability existsthat operator timing will be off and thus reproducibility compromised.Therefore, an inmunoassay apparatus for use with the immunoassaysdiscussed above should have the ability to manipulate one or morecapillary tubes and a sample tray, to control the flow of and housefluids, to carry out a substantial portion of the steps of theimmunoassay, including the step of detecting the and analyzing theflorescent signal. The ability to conduct a plurality of immunoassays ona plurality of samples also increases efficiency. Furthermore,quantitative analysis is possible with an integrated, semi-automated,immunoassay.

Another aspect of the present invention, therefore, is an apparatus fordetermining the presence of at least one analyte in a sample, whichapparatus comprises a reservoir, a means to control the flow of fluidcontained within the reservoir or in a sample tray, a first section toattach the cartridge of the present invention and similarly the sampletray of the present invention, a means to dry the capillary tubes, afluorometer to measure the level of fluorescence, and a means to analyzethe resulting signal and report a qualitative or semi-quantitativeresult and optionally a quantitative result.

The apparatus can cooperate with the cartridge and sample traycombination of the present invention and certain variations thereof. Ina typical immunoassay using the cartridge and sample tray combination incooperation with the apparatus of the present invention, a sample isadded to one or more wells in the sample tray. The sample tray is placedon the apparatus and a magnetic mixer located therein agitates the metalwashers of the sample tray to mix the contents of the well comprising afluorescently-labeled conjugate reagent and the sample so as to permitthe analyte in the sample to bind to the fluorescently-labeledconjugate. After the sample and reagent are properly mixed, theapparatus positions the sample tray under the cartridge located thereonand draws the mixture into one or more capillary tubes, e.g., viasuction. The mixture is incubated in the capillary tubes for asufficient time for a suitable amount of fluorescently-labeled conjugateto bind to the capture binding member of the substrate, on the surfaceof the capillary tube. After incubation, the apparatus passes a washsolution through the capillary tube to remove any unbound material. Theresulting waste fluid is emptied into the reservoir of the sample trayby the apparatus. The apparatus then notifies the operator to positionthe cartridge on the centrifuge to spin dry the capillary tubes.

The apparatus then positions the cartridge proximate to a fluorometer.The capillary tubes are exposed to an electromagnetic signal through anexposure opening located on the cartridge and the emitted fluorescenceis subsequently detected by a fluorescence detector. The detected signalcan be processed for qualitative, semi-quantitative, or quantitativeanalysis depending on the controls used, and on the analysis softwareemployed.

The level of quantitation possible using the apparatus with the devicesof the present invention depends on the affinity of the capture bindingmember as previously discussed, detector sensitivity, mathematics usedto analyze the signal, and whether standards and/or controls are usedand if so on what kinds of standards and/or controls. Generally,affinity of about 10⁶ L/mol can provide sensitivity in the parts permillion range and affinity of about 10⁹ L/mol can provide sensitivity inthe parts per billion range.

The most basic form of quantitation is the determination of the presenceof an analyte. For this to occur, the concentration of analyte in thesample must be above some lower limit of quantitation for theimmunoassay. Generally, a semi-quantitative result is reported. A barcode that accompanies the cartridge and sample tray kit containsinformation regarding the level of fluorescence that corresponds to theboundary between a pass an a fail as indicated by acceptance levels suchas the safe/tolerance levels shown in Table I. Typically, each lot ofreagent will have a different associated critical level of fluorescencedue to, among other things, variations in the binding affinity of thecapture binding member substrate. The apparatus will measure the levelof fluorescence and compare it to the pass/fail level for the specificimmunoassay corresponding to the concentration of interest.

More quantitative analysis is possible with the present invention. FIGS.16a-d to FIGS. 17a-b show plots of normalized fluorescence versusconcentration of analyte in parts per billion (ppb) for the β-lactamantibiotics used with the present invention. Normalized fluorescencecorresponds to the level of fluorescence emitted by a fluorescent labelbound to the surface of a capillary tube containing analyte as apercentage of the level of fluorescence emitted by a fluorescent labelbound to the surface of a capillary tube with no analyte, i.e. a blank.Since concentration is inversely proportional to the level offluorescence for a comparative assay, the curves formed by the pluralityof concentration points have a negative slope. If a sample is run on theapparatus, the resulting fluorescence signal can be compared against thefluorescence signal generated by a blank run in parallel with thesample. The resulting percentage can be plotted on the appropriate graphand a relative concentration of analyte in sample can be determined.

Generally, the apparatus will calculate a semi-quantitative result byusing a pass/no-pass level of fluorescence or a quantitative result byplotting a normalized level of fluorescence versus concentration, andcalculating a least-squares best fit of a line corresponding to thecurves. Thus a multilevel calibration curve can be used, whereinquantitative determination of the amount of analyte in a sample ispossible when such concentration is interpolated within the linear rangeof the best fit polynomial. Even greater quantitative results arepossible when standards are prepared to run in parallel with a sampleimmunoassay, wherein the same lot of fluorescent label is used for boththe sample conjugate and blank conjugate.

Since milk production is regulated by the FDA, immunoassays for β-lactamantibiotics are similarly regulated. The FDA typically requires thatpositive immunoassay results be confirmed. Consequently positive andnegative controls should be run with these immunoassays. One convenientmethod is the use of a control cartridge comprising passing, failing,and blank concentrations of analyte. This control cartridge can be runimmediately following a positive test result with the immunoassay.

DETAILED DESCRIPTION OF THE FIGURES

With a detailed description of the various aspects of the presentinvention provided, a detailed description of preferred embodiments ofthe devices and apparatus for use with the method for screening foranalytes as well as a detailed description of a preferred embodiment ofthe method as used with the devices and apparatus is now given. While adescription of preferred embodiments is given in considerable detail, itshould not be construed as limiting to those descriptions discussedhereinafter and variations thereof discussed hereafter. Other variationsof the described embodiments can occur to one of ordinary skill in theart that fall within the scope of the present invention.

With reference to terminology, it will be noted in the detaileddescription of the various aspects of this invention that portions ofthe devices are referred to as "top", "bottom", "obverse", "reverse","proximal" and "distal" portions. This is done wholly for convenienceand to relate the description to the diagrammatic representations in thedrawings. It will be appreciated that the devices can function in anyposition or orientation and it is within the scope of this invention tohave them do so.

FIG. 1 is a front perspective view of the devices of a preferredembodiment of the invention showing cooperation between the componentsduring use. The devices, which are preferably portable and disposable,comprise a cartridge 1, for sealingly holding four radially spaced apartantigen coated capillary tubes 2, where a reaction takes place; and asample tray 3 for containing sample, a fluorescently-labeled conjugatereagent, and other fluid, such as liquid waste; and for providingstorage of the cartridge 1. The sample tray 3, is positioned under thecartridge 1 by an apparatus discussed hereafter so as to permit thecartridge 1 to cooperatively draw fluid into each capillary tube 2 fromthe sample tray 3. The radial orientation of the capillary tubes 2functions to ensure that in cooperation with an exposure opening 7 eachcapillary tube 2 is presented to a signal detection means, discussedhereafter, at the same angle and to permit each capillary tube 2 to besubmitted to centrifugal force parallel to its longitudinal axis whilesecured within the cartridge.

FIG. 2 is a perspective view of the devices of a preferred embodiment ofthe invention showing cooperation between the components of the devicesduring storage when not in use. The cartridge 1 slides into a pair ofretaining shelves 83,84 discussed hereafter and snaps in place so as tobe secured by the retaining shelves 83,84 and a pair of fastening clips75, 76 discussed hereafter (also see FIG. 10).

FIG. 3a to FIG. 3h show the fully assembled cartridge 1 fromperspectives showing all sides of the cartridge 1 and particularlyshowing details of the various design features.

FIG. 4 is an exploded view of the devices of a preferred embodiment ofthe invention showing cooperation among the elements of the cartridge 1and sample tray 3. The cartridge 1 comprises a frame 4 and a holder 5for securing the capillary tubes 2 within the frame 4. The frame 4comprises a rigid substantially flat rectangular body preferably made ofpolystyrene, four radially oriented passageways 6 for containing fourglass capillary tubes 2 preferably coated with antigen (so as to conducta competitive assay), an exposure opening 7 for permitting a beam oflight to contact the capillary tubes 2 when secured within the cartridge1, and four protective tabs 8 for protection of the tips of thecapillary tubes 2.

FIG. 5 and FIG. 6 show perspective views of what can be referred to asthe reverse side of the frame 4. In a preferred embodiment shown, theframe forms a substantially flat and hollow rectangle about 4centimeters to about 10 centimeters, preferably about 5.6 centimeterslong along a side 9 and a wall 10; about 1 centimeter to about 8centimeters wide, preferably about 2.7 centimeters wide along two sidewalls 11, 12; and about 2 millimeters to about 1.3 centimeters high,preferably about 8 millimeters high along the three walls 10, 11, 12.The four passageways 6 originate from a rectangular protruding segment13 extending from the wall 10. The protruding segment 13 preferablyextends about 2 millimeters from the long wall 10 along the horizontalplane of the frame 4 as shown in FIG. 5 and is the same width as thelong wall 10, about 3.7 centimeters long. Located on each length wiseedge of the protruding segment 13 are three clips 14 for securelyattaching the holder 5 discussed hereafter to the protruding segment 13.Extending perpendicularly from the protruding segment 13 are two guidepins 15, 16, a first guide pin 15 and a segmented second guide pin 16,each for guiding a receptacle 35 and cap 36 discussed hereafter intoplace on the frame 4. The first guide pin 15 has an external diameterbetween 2 millimeters and 5 millimeters, preferably about 3 millimeters;and is about 1.5 millimeter to about 5 millimeters long or of asufficient length so that the end of the first guide pin 15 is flushwith the top of the cap 36, when the two are cooperatively engaged. Thesegmented second guide pin 16 has essentially the same length as thefirst guide pin 15 and an external diameter similar to that of the firstguide pin 15 at the base of the segmented second guide pin 16 and up toa ledge 17 located around the perimeter of the segmented second guidepin 16 at about the halfway point along the longitudinal axis of thesegmented second guide pin 16. From the ledge 17 to the end of thesegmented second guide pin 16, the external diameter of the segmentedsecond guide pin 16 is between about 1.5 millimeters to about 4.5millimeters, preferably about 2.5 millimeters. In any case of thispreferred embodiment, the segmented second guide pin 16 should have aledge 17 that defines two distinct external diameters that arecomplementary to the internal diameter of a corresponding segmentedguide shaft 50 discussed hereafter located on the cap 36. The functionof the different designs of the two guide pins 15, 16, is to permit onlyone orientation of engagement with the cap 36.

As shown in FIG. 6, along the 8 millimeter wide side of the protrudingsegment 13 are four equally spaced openings 18, each about 5 millimetersin diameter, for receiving the capillary tubes 2 and receptacle 35combination discussed hereafter. The openings 18 lead into eachpassageway 6 which comprises a receiving chamber 19 and a narrow shaft20 shown in FIG. 5 and FIG. 6. For the purpose of description, thepassageways can be divided into two pairs, an inner pair and an outerpair. The axis of the inner pair extend radially toward the long side 9preferably at about a 5 degree angle from an imaginary lineperpendicular to the center of the plane defined by the long wall 10.The axis of the outer pair extend radially toward the long side 9preferably at about an 18 degree angle from an imaginary lineperpendicular to the center of the plane defined by the long wall 10.The angles at which the capillary tubes 2 will extend from an imaginaryline perpendicular to the center of the plane defined from the long wall10 depend on the dimensions of the cartridge 1, but should be such thatthe capillary tubes 2 are radially orientated so as to ensure consistentexposure of each capillary tube 2 to a signal detection means, discussedhereafter, when the cartridge 1 is placed in a circular centrifuge, alsodiscussed hereafter, and to provide uniform centrifugal force to eachtube. That is, when an imaginary line is drawn along the longitudinalaxis of each capillary tube 2 when positioned in the cartridge 1, oneend of each of the lines should converge to a central point and the arcformed by the combination of the other ends of the lines should formpart of a circle. The receiving chambers 19 of the inner pair follow theaxis angle, are of the same diameter as the openings 18 or are slightlytapered, and are preferably about 4.5 millimeters deep. The receivingchambers 19 of the outer pair also follow the axis angle, are of thesame diameter as the openings 18 or are slightly tapered, and arepreferably about 6.5 millimeters deep. The function of the differentdepths of the receiving chambers 19 is to cooperatively engage thereceptacle discussed hereafter. The inner receiving chamber 19 and shaft20 combinations are about 1.5 centimeters to about 2 centimeters,preferably about 1.75 centimeters long, and the outer receiving chamber19 and shaft 20 combinations are about 1.3 centimeters to about 1.8centimeters, preferably about 1.6 centimeters long. The lengths of thecombinations of the inner and outer receiving chambers 19 and narrowshafts 20 are determined by the radial orientation of the passageways 6and a curve formed by an exposure opening 7, which curve functions toensure that each radially oriented capillary tube 2 positioned withinthe frame 4 is equally exposed to a signal detection means discussedhereafter when the cartridge 1 is positioned in a centrifuge discussedhereafter. Each shaft 20 is of a thickness sufficient for slidinglyfitting a capillary tube 2 therein. Each receiving chamber 19 and shaft20 combination is cross-sectionally open along the top plane of theframe 4. This results in a cross-sectional parabolic shape for the shaft20 that is about 5 millimeters deep. The cross-sectional cut is abyproduct of the design of a tool used to manufacture the frame 4.However, the cross-sectional cut of the shaft 20 also functions toexpose any damage to a capillary tube 2 held therein. At the end of theshafts 20 are openings 21, as shown in FIG. 5, through which thecapillary tubes 2 pass for presentation to the exposure opening 7. Asdiscussed previously, the exposure opening 7 forms a curve thatcorresponds to the radial orientation of the capillary tubes 2 so as toensure equal exposure of the capillary tubes 2 to a signal detectionmeans discussed hereafter. The exposure opening 7 is of a width suchthat preferably about an 8 millimeter portion of each capillary tube 2is exposed along the curve formed by the exposure opening 7 when securedwithin the frame 4 by the holder 5 discussed hereafter. The long side 9comprises a lip preferably about 1.5 millimeters deep and preferablyabout 3.2 millimeters high wherein the `L` formed by the lip facestowards the reverse side of the frame 4. The dimensions of the lip aresuch that the long side 9 is provided sufficient structural support tomake it substantially rigid. In this preferred embodiment, the lipextends into the frame 4 on either end about 3.2 millimeters to a curvedend of the two side walls 11, 12. At the top of the curved ends of eachof the two side walls 11, 12, is a semicircular resting ridge 22, 23,which extends preferably about 1.6 millimeters from the edge of the twoside walls 11, 12 along the same plane. The resting ridges 22, 23function to ensure that the cartridge 1 does not rock when laid on aflat surface with the reverse side of the frame 4 facing toward thesurface. The function of the resting ridges 22, 23 is necessary toconsistently orient the capillary tubes 2 at a proper angle to a signaldetection means when the cartridge 1 containing the capillary tubes 2 isinserted into a centrifuge, discussed hereafter, which permits exposureto the signal detection means discussed hereafter. Along the long side 9are four openings 24 for receiving the distal ends of the capillarytubes 2 and extending from the long side 9 at each receiving opening 24are the tabs 8 for protecting the tips of the capillary tubes 2. Eachtab 8 extends preferably about 3.2 millimeters from the long side 9 atthe same angle as the corresponding capillary tube 2, which extendsthrough the receiving opening 24 on the long side 9 such that the tip ofeach tab 8 is substantially flush with the tip of each capillary tube 2.As depicted in FIG. 4, the front of the frame 4 has a bevel 25, 26 ateither end of the exposure opening 7 positioned about 8 millimeters fromthe corners formed by the lip of the long side 9. Each bevel 25, 26 runsparallel to the long side 9, extends from each side wall 11,12 to theexposure opening 7, and slopes at approximately a 45 degree angle forabout 3 millimeters toward the obverse side of the frame 4 and towardthe long wall 10. The bevels 25, 26 function to permit the long side 9to support the distal ends of the capillary tubes via the receivingopenings 24 such that they are parallel to the plane of the frame 4while minimizing the use of plastic and additionally function to helpensure proper exposure of each capillary tube 2 to the signal generationand detection means discussed hereafter.

Turning again to FIG. 5 and FIG. 6, a pair of parabolically shapedfemale shafts 27, 28 can be seen. The female shafts 27, 28 comprise aparabolic element 29, 30, with the apex of the parabola pointing towardthe long wall 10 and a bendable clip 31, 32 with a lip 33, 34. Eachfemale shaft 27, 28, is preferably located about 6.4 millimeters fromthe long wall 10 and proximate to each side wall 11, 12 and extends awayfrom the reverse side of the frame 4 for about 8 millimeters at a 90degree angle. The bendable clips 31, 32 in combination with the lips 33,34 function as a locking mechanism to attach the cartridge 1 via thefemale shafts 27, 28 to an apparatus discussed hereafter designed foruse with the cartridge 1.

FIG. 4 shows the four capillary tubes 2 preferably coated with antigen.Each capillary tube 2 is preferably made of transparent silicon dioxideglass, more preferably borosilicate glass, and is about 10 millimetersto about 250 millimeters, preferably about 20 millimeters to about 150millimeters, and more preferably about 35 millimeters long with aninternal diameter of about 0.5 millimeters to about 1 millimeters,preferably about 0.65 millimeters. It should be noted that when thesample of a preferred embodiment is milk, an internal diameter of thecapillary tube 2 smaller than a preferred value of 0.65 millimeters canbe insufficient for the immunoassay, since aggregations of materials inthe milk can clog the capillary tubes 2 and interfere with fluid flow.The capillary tubes 2 frictionally fit into the receptacle discussedhereafter at one end and slidingly fit into the passageways 6 of theframe 4 on the other end.

FIG. 7 is an exploded view of the top side of the holder 5, and FIG. 8is an exploded view of the underside of the holder 5 for holding fourcapillary tubes 2 to be inserted into the frame 4. The holder 5comprises a receptacle 35 and a cap 36. The receptacle 35 is preferablymade of flexible Krayton® and comprises a flat rectangular support 37preferably about 8 millimeters wide by about 3 centimeters long andabout 1.6 millimeters thick, two guide openings 38, 39, and four collars40 connected to the support 37. The collars 40 function to frictionallyhold the capillary tubes 2 for insertion into the passageways 6 of theframe 4 with aid from the guide openings 38, 39 which slidingly engagethe guide pins 15, 16 located on the protruding segment 13 of the frame4. The diameter of the guide openings 38, 39 is about the same as theexternal diameter of the first guide pin 15 located on the protrudingsegment 13 of the frame 4, preferably about 3.4 millimeters. Each collar40 has a narrow passageway 41 therein with an internal diametersufficient to permit a capillary tube 2 to be inserted and held byfriction therein, a cone 42 that functions as a guide for inserting thecapillary tubes 2, and an opening 43 to permit insertion of thecapillary tubes 2. The combination receptacle 35 and capillary tubes 2slide into the frame 4 via the four openings 18 on the protrudingsegment 13. The collars 40 are of dimensions such that they canslidingly fit into the corresponding receiving chambers 19 of the frame4 as referenced previously. For the purpose of description, the collars40 can be divided into two pairs corresponding to the inner and outerpairs of the passageways 6 within the frame 4. The inner pair of collars40 are preferably about 4.8 millimeters long and extend radially awayfrom the center of the support 37 at the same angle as the correspondinginner pair of passageways 6 of the frame 4. The outer pair of collars 40are preferably about 6.4 millimeters long and extend radially away fromthe center of the support 37 at the same angle as the correspondingouter pair of passageways 6 of the frame 4. The varying lengths of thecollars function to extend the outer pair of radially oriented capillarytubes 2 such that the distal ends of all the capillary tubes 2 form aline parallel to the long side 9 of the frame 4 so that each capillarytube 2 penetrates each sample well discussed hereafter at an equivalentdepth. Each collar 40 is preferably about 3.2 millimeters in diameterand flares out at the cone 42 about 3 millimeters from the end to theopening 43 which has an external diameter of about 4.8 millimeters andan internal diameter of about 3.2 millimeters.

FIG. 7 and FIG. 8 show the rectangular cap 36, preferably made of rigidpolystyrene, for securing the receptacle 35 and capillary tubes 2combination in the frame 4. The cap 36 can be of any dimension suitableto cooperatively engage the receptacle 35 and protruding segment 13 ofthe frame 4, but preferably comprises two long walls 44, 45, about 3.8centimeters long by about 5 millimeters high; two short walls 46, 47about 4.8 millimeters high by about 9.5 millimeters wide at the topsloping down to about 8 millimeters wide at the bottom; and a top 48about 3.8 centimeters long by about a 9.5 millimeters wide. Locatedproximate to the short walls 46, 47 are two asymmetrical guide shafts49, 50, a first guide shaft 49 and a segmented second guide shaft 50,each for slidingly engaging the guide pins 15, 16, respectively, on theprotruding segment 13 of the frame 4 in combination with the guideopenings 38, 39 of the receptacle 35. The first guide shaft 49 extendsabove the top 48 to form a ridge about 0.5 millimeters to about 3millimeters, preferably about 1.5 millimeters high; has an internaldiameter of between about 2.1 millimeters to about 5.1 millimeters,preferably about 3.1 millimeters or an internal diameter such that itslidingly engages the first guide pin 15 located on the protrudingsegment 13 of the frame 4; and is preferably between about 1.5millimeters to about 5 millimeters deep or of a sufficient depth suchthat the top of the first guide pin 15 is flush with the top of thefirst guide shaft 49 when the two are cooperatively engaged asreferenced previously. The segmented second guide shaft 50 extends abovethe top 48 to form a ridge about 0.5 millimeters to about 3 millimeters,preferably about 1.5 millimeters high; has an internal diameter ofbetween about 2.1 millimeters to about 5.1 millimeters, preferably about3.1 millimeters or of a sufficient diameter to slidingly engage the baseof the segmented second guide pin 16 located on the protruding segment13 of the frame 4, and extending from the end facing the space formed bythe four walls 44, 45, 46, 47 to a ledge 51 located at about a half waypoint of the segmented second guide shaft 50. From the ledge 51 to thetop end of the segmented second guide shaft 50, the segmented secondguide shaft 50 has an internal diameter between about 1.7 millimeters toabout 4.7 millimeters, preferably about 2.7 millimeters or a sufficientinternal diameter to slidingly engage the upper portion of the segmentedsecond guide pin 16 located on the protruding segment 13 of the frame 4.The segmented second guide shaft 50 is preferably between about 1.5millimeters to about 5 millimeters deep or of a sufficient depth suchthat the top of the segmented second guide pin 16 located on theprotruding segment 13 of the frame 4 is flush with the top of thesegmented second guide shaft 50 when the two are cooperatively engagedas referenced previously. In any case, the segmented second guide shaft50 should have a ledge 51 that defines two distinct internal diameterssuch that the segmented second guide shaft 50 is complementary to thecorresponding segmented second guide pin 16 located on the protrudingsegment 13 of the frame 4 and can slidingly engage therein. The positionof the asymmetrical guide shafts 49, 50 is such that they properly alignwith the guide pins 15, 16 and guide openings 38, 39. The difference ofthe dimensions of the two asymmetrical guide shafts 49, 50, functions topermit only one orientation for engaging the holder 5 comprising the cap36 and receptacle 35 with the frame 4 as previously discussed. That is,the segmented second guide shaft 50 can only engage the segmented secondguide pin 16 located on the protruding segment 13 of the frame 4 andcannot engage the first guide pin 15 similarly located. This ensuresthat the opening of a single port 54, discussed hereafter, located onthe cap 36 faces away from the obverse side of the frame 4 when theholder 5 and the frame 4 are fully assembled to form the cartridge 5.Attached to the top 48 of the cap 36 is a trough 52 preferably about 2.2centimeters long by about 1.6 millimeters wide and preferably about 1.6millimeters deep and opens into the space created by the four walls 44,45, 46, 47. The trough 52 is designed such that when it contacts thesupport 37 of the receptacle 35, it forms a sealed chamber so that thecapillary tubes 2 are in fluid communication with each other. The sealis provided by an narrow oval ridge 53, shown in FIG. 8, that encirclesthe trough 52 about 2 millimeters from the edges of the trough 52 inthis preferred embodiment, but can be any appropriate distance aroundthe trough 52; and about 3 millimeters from the ends of the trough 52and presses against the support 37 of the receptacle 35. Attached to thetrough 52 at its center is a single port 54, shown in FIG. 7, with aninternal diameter preferably about 3.2 millimeters which can beconnected to a luer fitting discussed hereafter for pumping fluidthrough the chamber formed by the trough 52 and receptacle support 37.The single port 54 is about 8 millimeters long and is oriented such thatits passage runs parallel to the horizontal plane of the top 48 whileleading away from the trough 52 at a perpendicular angle as shown inFIG. 7. An opening 55 connecting the single port 54 to the trough 52 islocated at the center of the trough 52 as shown in FIG. 7. Eachlongitudinal wall 44, 45 of the cap 36 contains three equally spacedrectangular openings 56 which fit over the three equally spaced clips 14located on each of the lengthwise edges of the protruding segment 13located on the frame 4 as discussed previously. The cooperation betweenthe openings 56 and the clips 14 functions to secure the cap 36,receptacle 35, and capillary tubes 2 combination over the protrudingsegment 13 of the frame 4; with the aid of the guide pins 15, 16, theguide openings 38, 39, and guide shafts 49, 50; and with additional aidof the angle of the long cap walls 44, 45 formed by the short cap walls46, 47. The combination frame 4, capillary tubes 2, receptacle 35, andcap 36 therefore comprise the cartridge 1 in this preferred embodiment.

FIG. 9 and FIG. 10 are perspective views of a preferred embodiment ofthe sample tray 3 which is preferably made of polypropylene. The sampletray 3 comprises four equally spaced wells 57 for holding aliquots of afluorescently-labeled conjugate reagent and/or a sample, a reservoir 58for holding fluid, such as liquid waste; and a molded cartridge storagecompartment 59. The sample tray 3 forms a square preferably about 6centimeters on all sides. The wells 57 are supported by a shelf 60. Theshelf 60 extends from a wall 61 running the full width of the tray 3,and attached to two side walls 62, 63. The wall 61 from which the shelf61 extends is about 6 centimeters long and preferably about 9.5millimeters high. The two side walls 62, 63 are each preferably about2.5 centimeters long and preferably about 1.4 centimeters high. Theshelf 60 originates from the long wall 61 preferably about 1.6millimeters below the top edge of the wall 61 such that a lip 64 isformed to aid in retention of any escaped liquid. The shelf 60 extendsfrom the wall 61 at a slight downward angle that functions to guideescaped liquid to the reservoir 58 discussed hereafter and extends forabout 9.5 millimeters and attaches to a wall 67 perpendicular to itwhich drops from it to the floor 68 of the reservoir 58 discussedhereafter. Each well 57 located on the shelf 60 has a parabolic shapewith an internal diameter preferably about 8 millimeters and a depthpreferably about 4.7 millimeters. Held within at least one well 57 is ametallic object comprising a stainless steel washer 65 for mixing. Bypassing a varying magnetic field under the washer 65, a driedfluorescently-labeled conjugate reagent resting on the bottom of thewell 57 and optionally adhered to the bottom of the well 57, isreconstituted when a solution (optionally containing a sample) is addedto the well 57. About a 4.8 millimeter diameter retaining ridge 66 islocated along the opening to each well 57 and functions to prevent thewasher 65 from falling out of the well 57.

The reservoir 58 is defined by a wall 67 which drops about 5 centimetersfrom the edge of the shelf 60, as discussed previously, to a floor 68;the portions of the side walls 62, 63 extending from the shelf 60, and amolded center wall 69 which rises preferably about 1.3 centimeters fromthe floor 68 such that it is about flush with the tops of the side walls62, 63 and also forms part of the cartridge storage compartment 59discussed hereafter. The reservoir 58 functions to contain fluid,discussed previously, passed through the capillary tubes 2 via thesingle port 54 on the cap 36 of the cartridge 1. The volume of thereservoir 58 is defined by the length and height of the wall 67 anddistance from the wall 67 to the molded center wall 69 and should be ofsufficient dimensions to contain an appropriate multiple of the combinedvolume of fluid capable of being contained by all the capillary tubes 2contained within the cartridge 1.

FIG. 9 and FIG. 10 also show the molded cartridge storage compartment 59which forms part of the sample tray 3. The compartment 59 is designed toform a molded contour around the cartridge 1 and securely retain thecartridge 1 within the sample tray 3 for storage and transportation asshown in FIG. 2. The compartment 59 is defined on its periphery by themolded center wall 69, a rear wall 70 opposite the center wall, twopartial walls 71, 72, and a floor 73. The center wall 69 is molded toconform to the dimensions defined by the portion of the cartridge 1comprising the long wall 10 of the frame 4, and the cap 36 secured onthe protruding segment 13 of the frame 4 as shown in FIG. 2. At thecenter of the center wall 69 is a curved section 74 forming a halfcircle which curves toward the shelf 60 and is of such dimensions thatit can slidingly receive the portion of the cartridge 1 defined by thesingle port 54 wherein the arc defined by the curve of the curvedsection 74 is substantially similar to the arc of the curve defined bythe single port 54 such that the curved section 74 substantially forms auniform contact with the periphery of the single port 54. The centerwall 69 extends linearly on either side of the curved section 74, bendsperpendicularly on either side towards the rear wall 70 to form a moldthat encases the cap 36 of the cartridge 1, and bends againperpendicularly on either side towards and attaches to the two sidewalls 62 and 63. In this manner the center wall 69 forms a mold thatsubstantially forms a uniform contact with the surfaces of the cartridgeformed by the side of the cartridge 1 containing the holder 5. Locatednear to either edge of the curved section 74 and facing towards the rearwall 70 are a pair of fastening clips 75, 76 for securely holding thecartridge 1 within the storage compartment 59 in cooperation with thepair of retaining shelves 83, 84 discussed hereafter. The fasteningclips 75, 76 are positioned at a height above the floor 73 such thatthey will engage the edge of the cap 36 comprising the corner formed bythe front wall 44 and top 48 of the cap 36 of the cartridge 1 when thecartridge is positioned within the compartment 59 of the sample tray 3with the reverse side facing the floor 73 as shown in FIG. 2. The twopartial walls 71, 72 extend from the rear wall 70 for about 1 centimeterand are of the same height as the rear wall 70. At each corner formed bythe partial walls 71, 72 and the rear wall 70 is a column 77, 78extending from the floor 73 to the edge of the walls 70, 71, 72. Eachcolumn 77, 78 is adjacent to each corner at two sides, forms a side 79,80 extending perpendicularly away from each of the partial walls 71, 72and towards each other for a distance about the same as the distancefrom each of the corners formed by side 9 on the frame 4 of thecartridge 1 to the base of the outer pair of protective tabs 8, andangles towards the rear wall 70 at an angle corresponding to the angleof the outer pair of protective tabs 8 to form an angled side 81, 82.The columns 77, 78 function to hold the side of the cartridge 1comprising the long side 9 of the frame 4 and protective tabs 8extending from the long side 9, whereby the tips of the protective tabs8 rest against the rear wall 70 and the corners of the long side 9 ofthe cartridge 1 rest against the column sides 79, 80 and the partialsides 71, 72 as shown in FIG. 2. Extending from each of the cornersformed by the partial walls 71, 72 and the column sides 79, 80 is anarrow retaining shelf 83, 84 that forms a square with sides of equalwidth to the adjacent column side 79, 80 and positioned at a heightabove the floor 73 such that the obverse planer portion adjacent to thecorners nearest the protective tabs 8 of the cartridge 1 rest againstthe underside of each of the retaining shelves 83, 84 so as to securethe cartridge 1 within the storage compartment 59 of the sample tray 3as discussed previously and as shown in FIG. 2. Connecting the base ofeach of the corners formed by central wall 69 and short walls 62, 63 tothe ends of the partial walls 71, 72 is a curved ledge 85, 86 thatfunctions to aid removal of the cartridge 1 from the storage compartment59. The curved ledges 85, 86 curve inward to form an arc complementaryto the shape of human finger and rises above the floor 73 to a heightsufficient for portions of the edges of the side walls 11, 12 of theframe 4 of the cartridge 1 to rest against the curved shelves 85, 86when secured within the storage compartment 59 of the sample tray 3 asshown in FIG. 2. Extending inward from the rear wall 70 and alongconverging axis are three braces 87, 88, 89 for providing support forthe cartridge 1 while positioned in the storage compartment 59 of thesample tray 3 by bracing against the obverse junctions of the receivingchambers 19 of the frame 4 and the edge of the lip formed by the longside 9 of the frame 4. The braces 87, 88, 89 are of dimensions thatpermit distal contact of the receiving chamber 19 junctions whileproviding a path for the narrow shafts 20. In a preferred embodimentshown, the three braces 87, 88, 89 comprise three narrow walls, but canalso comprise three stubs along the rear wall 70 and three stubs tocontact the junctions previously described. Along the outer edges ofeach of the external walls 62, 63, 70, 71, 72 is a lip 90 about 3millimeters wide that aids the physical support of the sample tray 3.

FIG. 11 is a perspective view of another embodiment of a sample tray103. The sample tray 103 comprises four equally spaced wells 157supported on a shelf 160 for holding aliquots of a fluorescently-labeledconjugate reagent and/or a sample as in a preferred embodiment discussedabove, and a reservoir 158, for holding fluid, such as liquid waste;defined by the shelf 160, three walls 162, 163, 170, and a floor 173.The sample tray 103 forms a square preferably of the same dimensions asa preferred embodiment of the tray 3 discussed previously. The two walls162, 163 that run perpendicular to the shelf 160 are preferably about1.4 centimeters high and the wall 170 opposite the shelf 160 ispreferably about 9.5 millimeters high. Extending along the top edge ofwalls 162, 163, 170 is about a 3 millimeter lip 190 which aids physicalsupport of the sample tray 103. The four wells 157 are located along theshelf 160 in an equally spaced manner, are preferably of the samedimensions as the wells 57 of a preferred embodiment previouslydiscussed. The wells 157 preferably contain a stainless steel washer ordisc 165 for mixing a dried reagent resting on the bottom of the well157, optionally, the reagent can be coated on the washer. The reagent isreconstituted when a solution optionally containing a sample is added tothe well 157. Wells contain a retaining ridge 166 similar to theretaining ridge 66 of a preferred embodiment previously discussed. Theshelf 160 is preferably of the same dimensions as the shelf 60 of apreferred embodiment discussed above with a wall 167 extending to thefloor 173 preferably similar to the corresponding wall 67 of a preferredembodiment discussed above.

In this alternative embodiment depicted in FIG. 11, the reservoir 159 isdefined by the three walls 162, 163, 170, the shelf 167, and the floor173 and, as stated above, similarly functions to contain fluid, such asliquid waste, as does the reservoir 59 of a preferred embodimentdiscussed above. Located preferably about 3.2 centimeters from the edgeof the shelf 160 is a bevel 195 extending across the reservoir 159 fromone side to the other 162, 163. The bevel 195 is preferably about 4.7millimeters wide and slopes at about a 45 degree angle to form a secondshelf 196 about 3.2 millimeters above the floor 173 and about 1centimeter deep. The bevel 195 functions to prevent fluid, typicallyliquid, from being splashed against the rear wall 170 when the sampletray 103 is tipped. The cartridge 1 can also be stored in thisembodiment of the sample tray 103 by laying it flat on the bottom 173such that the protective tabs 8 face the rear wall 170. The cartridge 1can be secured within the sample tray 103 by a plastic cover or bysimilar means.

FIG. 12 is a perspective view of a preferred embodiment of the apparatus200 for use with the cartridge 1 and sample tray 3 combination of thepresent invention. FIG. 13 is a transparent view of a preferredembodiment of the apparatus 200 showing the critical internalcomponents. Referring to FIGS. 12 and 13, the apparatus 200 comprises aprep station 201, a centrifuge 202 for drying the capillary tubes 2, asignal generation and detection station 203 for measuring a signal, analphanumeric keypad controller 204 for permitting interaction between anoperator and the apparatus 200, a liquid crystal display (LCD) 205 fordisplaying input and output messages and analytical results produced bya computing device discussed hereafter, and a printer 206 for printing aresult.

Referring again to FIG. 12 and FIG. 13 the prep station 201 comprises areservoir 207 for containing a fluid, a conduit 208 for transporting thefluid to a port 209, a syringe 210 for drawing sample to the port 209and for pumping fluid from the reservoir 207 through the port 209; afirst section comprising a previously referenced luer fitting 211 forattaching the single port 54 of the cartridge 1 to and for permittingfluid communication with the port 209 of the apparatus 200; a secondsection comprising a tray holder 212 for holding the sample tray 3, anda magnetic mixer 213 for mixing fluid in the wells 57. Magnetic mixer213 is positioned under the tray holder 212 in such a manner as to alignwith the wells 57 of the sample tray 3 when the sample tray 3 ispositioned in the tray holder 212. The centrifuge 202 is housed in aprotective compartment 214 and comprises a disc capable of securelyengaging the cartridge 1. The electromagnetic signal generation anddetection station 203 comprises a signal generation means 215, such aslaser or tungsten lamp suitable to emit an electromagnetic signal at anappropriate wavelength to cause a fluorescing compound in thesample-reagent mixture to become excited and fluoresce, and a signaldetection means 216, such as a photon detector for detectingfluorescence, which photon detector can comprise a photomultiplier,phototube, photocell, or silicon diode, and preferably comprises asilicon diode.

FIG. 14a is a block diagram of the apparatus of a preferred embodimentof the invention showing cooperation among the electronic parts and FIG.14b is a similar block diagram of the apparatus showing designationsknown to those of ordinary skill in the art of the various components. Alogic and motor control printed circuit assembly (PCA) 300, hereafterreferred to as a control PCA, functions to control and interface withthe various components of the apparatus 200. A 40 wire ribbon cable 301connected to two 40 position ribbon headers 302, 303 functions toconnect the control PCA 300 to a prep station breakout PCA 304 forcontrolling the various components of the prep station 201.

The electrical components connected to the prep station PCA 304 comprisethe following:

a syringe motor 305 for actuating and accurately controlling movement ofthe syringe 210, which syringe motor 305 is connected to the prepstation PCA 304 via a first 8 position amplitude modulator 306;

a mixer motor 307 for actuating the magnetic mixer 213, which mixermotor 307 is connected to the prep station PCA 304 via a second 8position amplitude modulator 308;

a lift motor 309 for moving the tray holder 212 to properly position thesample tray 3 under the cartridge 1, which lift motor 309 is connectedto the prep station PCA 304 via a third 8 position amplitude modulator310;

a valve 311 for controlling the direction of flow of the fluid containedin the reservoir 207, which valve 311 is connected to the prep stationPCA 304 by 6 position amplitude modulator 312;

a syringe sensor 313 for monitoring the position of the syringe 210,which syringe sensor 313 is connected to the prep station PCA 304 via afourth 8 position amplitude modulator 314; a mixer sensor 315 formonitoring the position of the magnetic mixer 213, which mixer sensor315 is connected to the prep station PCA 304 via a fifth 8 positionamplitude modulator 316; and

a lift sensor 317 for monitoring the position of the lift motor 309,which lift sensor 317 is connected to the prep station PCA 304 via asixth 8 position amplitude modulator 318.

A 16 position amplitude modulator 319 connects the control PCA 300 tothree additional PCA cards, a laser PCA 320 for controlling theoperation of the signal generation means, which laser PCA 320 isconnected to the control PCA 300 via a 6 position molex pocket header321; a preamplifier PCA 322 for amplifying a photogenerated signalbefore processing by the control PCA 300, which preamplifier PCA 322 isconnected to the control PCA 300 via a 5 position molex pocket header323; and an barcode PCA 324 for interfacing with a commerciallyavailable barcode wand discussed hereafter, which barcode PCA 324 isconnected to the control PCA 300 via a 4 position molex pocket header325 connect. An 18 position amplitude modulator 326 connects the variouselectrical components of the centrifuge 202 to the control PCA 300. Theelectrical components of the centrifuge 202 comprise a rotor motor 327for rotating the centrifuge 202, a rotor door sensor 328 for detectingthe opening of the door of the protective compartment 214, and a rotorhome sensor 329 for aiding with determination of the position of thecartridge 1 while in the centrifuge 202 so as to permit properpresentation of the cartridge 1 to the signal generation and detectionstation 203. A 50 wire ribbon cable 330, connected to two 50 positionribbon headers 331, 332, connects a peripheral adapter PCA 333 thatfunctions to interface with the alphanumeric keypad 204, the printer206, the liquid crystal display 205, and an audio buzzer 334 forproviding audible feedback to the operator about the functional statusof the apparatus 200. A panel connector PCA 335 located at the rear ofthe apparatus 200 interfaces with an external DC power input connector336, an RS-232 port 337, and a barcode wand input connector 338. Thepanel connector PCA 335 is connected to the control PCA 300 via a 10wire ribbon cable 339 connected at each end to a 10 position ribbonheader 340, 341; and via a power cable 342. Main power to the apparatus200 is supplied via serial cooperation among a 110 vac input plug 343, apower entry module 344, a signal transformer 345, and an AC input cable346 as shown in FIG. 14a and 14b. Two 2 position mini-fit molexconnectors 347 connect the AC power cord 346 and the DC power cord 342to the control PCA 300. The automated non-operator dependent portion ofthe apparatus 200 system software is provided by an erasableprogrammable read only memory (EPROM) module 348 connected to thecontrol PCA 300 via a 30 position edge connector 349.

A Typical Immunoassay Using preferred embodiments of the PresentInvention

FIG. 15 is a simplified representation of the solid-phase fluorescenceimmunoassay (SPFIA) of a preferred embodiment of the present invention.In a typical immunoassay using a preferred embodiment of the presentinvention, the cartridge 1 and sample tray 3 are placed on the prepstation 201 by the operator as shown in FIG. 12, then 100 microliters ofa sample possibly containing an analyte of interest, such as milksuspected of containing β-lactam antibiotics, is added to one or morewells 57 located in the sample tray 3 which contain a dried stabilizedreagent comprising an antibody to the analyte of interest, whichantibody is conjugated with a highly fluorescent label, such as acyanine dye (Cy-5®), fluorescein isothiocyanate (FITC), rhodamine, TexasRed, phycoerythrin, and allophycoerythrin or any other compound with asufficient quantum yield or efficiency to produce appropriatefluorescence and that will not interfere with any reactions of theimmunoassay; or with a bio-luminescent compound. A barcode, providedwith the cartridge 1 and sample tray 3 combination kit, is then scannedusing a standard commercially available barcode wand associated with thebarcode wand input connector 338 to provide calibration information tothe apparatus 200. For example, the barcode can contain the pass/failthreshold level of fluorescence for a particular immunoassay. Theoperator then inputs additional information via the alphanumeric keypadcontroller 204 and subsequently starts the apparatus 200. The sample andfluorescently-labeled conjugate reagent are then mixed while in the well57 by the application of a magnetic field provided by the magnetic mixer213 located under the tray holder 212 of the apparatus 200, such thatthe metallic washer 65 flips, rotates or agitates to appropriately mixthe sample and conjugate reagent combination for a sufficient time forthe fluorescently-labeled conjugate and the analyte to bind. Thecartridge 1 is connected via the single port 54 on the cap 36 of thecartridge 1 to the luer fitting 211 of the prep station 201 of theapparatus 200 and positioned above the sample tray 3 with the obverseside of the cartridge 1 facing the reservoir 58. Within the cartridge 1each capillary tube 2 has at least a portion of the internal surfacecoated with antigen analogous to or mimetic of the analyte of interest.The sample tray holder 212, and thereby the sample tray 3, is lifted bythe lift motor 309 so that the tips of the capillary tubes 2 contacteach sample and fluorescently-labeled conjugate reagent mixture in thewells 57. The sample tray 1 is oriented in a manner which permits thecartridge 1 to be positioned such that the capillary tubes can penetratethe sample and fluorescently-labeled conjugate reagent mixture at asufficient depth and at a sufficient angle to draw a sufficient amountof the sample and fluorescently-labeled conjugate reagent mixture intoeach capillary tube 2. The sample and fluorescently-labeled conjugatereagent mixture is then drawn into the capillary tube via capillaryaction and suction applied by the syringe 210 to the single port 54 onthe cap 36 of the cartridge 1, and is then allowed to incubate for aperiod of time in one or more capillary tubes 2 during which apercentage of fluorescently-labeled conjugate not bound to the analyte,will bind to the antigen comprised by the substrate comprising thecapillary tube 2 walls. If there is a relatively large amount of analytein the sample, very little fluorescently-labeled conjugate will bind tothe substrate of the capillary tube 2 walls and if there is a relativelysmall amount of analyte in the sample a large amount offluorescently-labeled conjugate will bind to the substrate of capillarytube 2 walls.

After the incubation period, a liquid residing in the reservoir 207 ofthe apparatus 200 is pumped by the syringe 210 through each capillarytube 2 via the luer fitting 211 and the single port 54 located on thecap 36. The liquid then enters the chamber created by the trough 52 andreceptacle support 37 and subsequently enters each capillary tube 2. Theliquid stops the reaction and washes excess fluid out of each capillarytube 2. Prior to or simultaneous with the pumping of the liquid intoeach capillary tube 2, the lift motor 309 orients the sample tray holder212, and thereby the sample tray 3, such that the capillary tubes 2 arepositioned over the reservoir 58 within the sample tray 3, so that thefluid pumped out of each capillary tube 2 is expelled into the wastereservoir 58 of the sample tray 3. The sample tray 3 is then discardedand the cartridge 1 is inserted into and dried by the centrifuge 202located on the apparatus 200 for use with the cartridge 1 and sampletray 3 combination. After drying and while in the centrifuge 202, thecartridge 1 is positioned in front of the signal generation means 215,such that the exposure opening 7 of the frame 4 permits the signalgeneration means 215 to emit a signal that substantially penetrates thecapillary tube 2. The signal detection means 216 positioned adjacent tothe signal generation means 215 then detects any fluorescence emitted bythe bound conjugate containing the fluorescent label. This signal isthen processed by the computing portion of the apparatus 200 and thepresence of analyte, alternatively the amount of analyte present isdetermined where analyte concentration is inversely proportional to thelevel of fluorescence for this preferred embodiment (a competitiveassay). FIGS. 16a-d to FIGS. 17a-b show plots of normalized fluorescenceversus concentration of six β-lactam antibiotics in parts per billion atdifferent levels of concentration. A least squares polynomial fit of thecurves generated by the multiple points can be used by the apparatus 200to provide a quantitative result, wherein the computing portion of theapparatus 200 interpolates the amount of analyte in the sample byplotting the observed level of fluorescence on the appropriatecalibration line for the particular analyte and identifying thecorresponding concentration for that level of fluorescence.

It should be noted that in a presently preferred embodiment of thepresent invention, the operator only carries out the steps of placingthe sample tray 3 in the sample tray holder 212, adding sample to thesample tray 3, optionally scanning the barcode, starting the apparatus200, moving the cartridge 1 to the centrifuge 202, and discarding thecartridge 1 and sample tray 3. The apparatus 200, performs all the othersteps of the immunoassay discussed above.

Alternative Embodiments and Variations of preferred embodiments

Although the present invention has been described in considerable detailwith reference to a preferred embodiments thereof, other embodiments arepossible. For example variations of either one or all of the immunoassaychemistry used, the cartridge and sample tray design, and the design ofthe apparatus for use with the cartridge and tray combination arepossible within the scope of the present invention.

A preferred variation for the immunoassay chemistry is simply to reversethe label and binding member in the competitive fluorescence immunoassayof a preferred embodiment of the invention. That is, thefluorescently-labeled conjugate reagent can contain an antigen analogousto or mimetic of the analyte, wherein the antigen is conjugated with thefluorescent label; and the antibody to the analyte is coated onto thesurface of the capillary tubes. In this case, the fluorescently-labeledantigen conjugate competes with the analyte antigen for binding sites onthe antibody coated on the surface of the capillary tubes. As in thecompetitive fluorescence immunoassay of a preferred embodiment of thepresent invention, the level of fluorescence will be inverselyproportional to the amount of analyte in the sample.

In addition to the competitive fluorescence immunoassay of preferredembodiments described above, a non-competitive sandwich fluorescenceimmunoassay can also be used. In this case, the sample is mixed with afluorescently-labeled conjugate reagent and drawn into the capillarytube. The analyte binds to the conjugate and to a capture binding memberon the substrate which comprises a surface of the capillary tube. Thecapillary tube is washed, optionally dried, and exposed to a signalgeneration means and signal detection means to measure the level offluorescence. In a sandwich immunoassay such as this, the amount offluorescence is directly proportional to the amount of analyte in thesample.

While an immunoassay described above as a preferred embodiment is acompetitive inhibition immunoassay employing a fluorescent label, otherimmunoassay methods can also be employed. These include ELISA sandwichimmunoassays wherein an analyte sample is mixed with free antibody tothe antigen and subsequently washed and further mixed with an enzymelinked antibody specific to a different epitope on the analyte. Any freeantibody is then washed away and an enzyme substrate is added to complexwith the enzyme mixture. Detection of the analyte can then beeffectuated calorimetrically or via other means whereby the level of thesignal is positively correlated to the concentration of analytedetected.

As noted above, variations on the immunoassay can include differentlabels, such as enzymes for use in a non-competitive immunoassay.Similarly, an enzyme label can be used in a competitive immunoassaywhereby detection of a signal generated by a UV lamp occurs via a UVabsorbance detector and the absorbance reading is negatively correlatedwith the concentration of analyte in the sample. Alternatively RIAimmunoassays known to those of ordinary skill in the art can be usedwith an appropriate embodiment of the present invention.

The dimensions of the cartridge and sample tray can vary from thatalready described depending on the particular application and use. Allthat is required are dimensions appropriate for use with an apparatuscomprising any analytical instrument sufficient to effectuate the chosenimmunoassay. For example, these dimensions include a length of capillarytube sufficient to contain an amount of a substrate and sample mixture,a cartridge comprising a region suitable to expose at least a portion ofeach tube to an emitted signal, and a sample tray with wells thereinsufficient to contain sample and reagent solution. Furthermore, areservoir can optionally be included with a waste disposal meanscomprising a vacuum system connected to the cartridge for instance,wherein the evacuation product of the capillary tube contents arecontained in a separate container or chamber.

In many cases, the apparatus, the cartridge or tube design will dependon the type of immunoassay employed, instrumentation with which thesubject interacts, and conditions under which the subject is used.Immunoassays that can be adapted for use with the present invention orvariations thereof include ELISA, RIA, and FIA as discussed previously,but can also include other bioassays, including but not limited tovarious other immunoassays utilizing different labels, such as enzymes,isotopes, fluorescent and bio-luminescent compounds, physicalconstructs, reactants; and other bioassays appropriate for use incapillary tubes. Instrumentation can include custom or commerciallyavailable analytical instruments including, but not limited, tospectrophotometers, chromatographs for collection fractions from theimmunoassay, counters, densitometers, diagnostic instruments, andforensic instruments; and other instruments necessary for use with aparticular embodiment of the present invention. Conditions for useinclude, but are not limited to, clinical laboratories, use in thefield, such as outdoors and/or in agricultural, dairy, and industrialsettings; and in research or crime laboratories. Alternative embodimentsof the present invention can thus be used to adapt to the above uses,conditions for use, and cooperation with other devices.

The cartridge can comprise substantially one piece with the coatedcapillary tubes frictionally sealed within the cartridge. Alternatively,the capillary tubes can be sealed within the cartridge by a means otherthan by friction, such as an adhesive sealant. The capillary tubes canbe spaced apart linearly rather than radially. In this case, dryingpreferably occurs via a means other than by centrifugation, such as byair drying, by purging with a gas, such as nitrogen; or by vacuum. Thecartridge can have a plurality of chambers each independently connectedto each capillary tube. In this case an even greater diversity ofimmunoassays can be carried out in each capillary tube, including theuse of different detectable events and detection means. Protection ofthe capillary tubes can also be accomplished by various means, such asuse of a hinged cover that can be optionally positioned over the tips ofthe capillary tubes and moved away from the capillary tubes when fluidis drawn or added to the capillary tubes, or the capillary tubes can beretractable into and out of the cartridge. The region of the cartridgefor exposing each capillary tube to a signal detection means can also bedesigned so that only one side of the cartridge is open since, in thecase of fluorescence immunoassays, the signal generation means andsignal detection means can be adjacent to each other. Additionally, aclear plastic cover can be located over the exposure opening 7 toprotect the capillary tubes from damage.

One embodiment of a one piece design is a cartridge wherein thecapillary tubes are linearly spaced apart within the cartridge with acentral opening for exposing them to a signal detection means. On oneside of the capillary tubes can be a receiving chamber for receiving asample, such as milk. A syringe on the other side of the capillary tubescan be used to draw sample into the capillary tubes. To conduct theimmunoassay sample in the receiving chamber is placed in contact withthe capillary tubes by passageways that connect to individual reagentchambers in fluid communication with each capillary tube. The reagentchambers can contain reagent such that backward engagement of thesyringe can draw sample into these chambers and stop to permit mixtureof sample and reagent in each chamber by magnetic or other meansdiscussed herein or known to those of ordinary skill in the art.Backward engagement of the syringe can then continue and be used to drawthe reagent sample mixture from the mixing/reagent chamber and into thecapillary tubes for incubation and an immunoassay reaction to occur. Thesame syringe can be used to push a fluid into the capillary tubes via apassage connected to the distal end of each capillary tube, wherein athird chamber containing wash fluid for instance, can be opened via avalve or puncture means such that forward engagement of the syringeforces the fluid into the passageway and through each capillary tube. Inthis embodiment a drying step would not be required.

The cartridge can also be of an entirely different shape. For example,the cartridge can comprise a disc wherein the capillary tubes are spacedradially throughout the disc and a sample can be added to chambers atthe periphery of the disc in such a manner that sample can be drawn intoeach capillary tube and expelled into the first chamber by pumped fluidadded to the tubes via a port at the center of the disc; or othervariations of a circular cartridge.

Similarly, a circular disc can be used wherein the sample is added to afirst chamber near the center of the disc. Upon application ofcentrifugal force, the sample, which can also react with a reagent inthe first chamber, can be drawn into capillary tubes or passagewaysradially spaced apart within the disc wherein one or more secondchambers at the periphery of the disc and fluidly attached to thecapillary tubes can collect excess fluid or provide for further reactionsteps. Variations of this embodiment can include additional capillarypassageways and/or chambers. Alternatively the cartridge can be shapedlike a pie wedge such that a plurality of cartridges can fit together toform a circle for drying in a centrifuge or for permitting reactions tooccur as previously described.

Another embodiment is a "Gatling gun" design wherein the capillary tubesare spaced apart along the outside of the longitudinal side of acylinder or by a disc or plurality of discs containing openings to holda plurality of spaced apart capillary tubes in a circular manner. Inthis case the cylinder can rotate to present a particular tube to asample source or to a fluid source for washing, adding reagent, or otherdesired fluid to each tube. The cylinder can also rotate to present acapillary tube to a signal generation and detection means.Alternatively, the cylinder can cooperate with a multiwelled circularsample tray for serial or simultaneous sample removal and/or washing andexpelling of fluid and even multiple signal generation and detectionmeans. The circularly spaced apart capillary tubes can encircle acentral syringe that is in fluid communication with the capillary tubesand draws fluid into the capillary tubes and/or advances fluid from thecapillary tubes into any chambers and/or reaction sites on the surfaceof the capillary tubes.

The cartridge can also comprise a substantially rectangular shape wherea plurality of capillary tubes are proportionately spaced apart andarranged in two rows; e.g. tubes are spaced in a cartridge that has anexposure opening on each side, which thereby permits simultaneousdetection of analyte in two or more samples. In this embodiment, twopairs of signal generation and signal detection means can besimultaneously used, wherein the cartridge is designed such that eachrow of capillary tubes can be simultaneously or independently contactedby signal generation and signal detection means when mounted on anappropriately designed apparatus, such as an analytical instrument.

The cartridge can also be made of different types of plastic such as,polyvinyl chloride, polyethylenes, polyurethanes, polystyrenes,polypropylenes, and other plastic materials. The capillary tubes canalso optionally be made of plastic such as polyvinyl chloride,polyurethanes, polystyrenes, polypropylenes, polyethylenes and otherplastic materials commonly used to form capillary tubes or tubing.Preferably, the chosen material not interfere with either the chemistryof the immunoassay or any analytes or products thereof, or the signalgeneration and detection means. Preferably the chosen material providesfor an inexpensive and therefore disposable cartridge design.

The sample tray can also vary from preferred and an alternativeembodiment described above. It can have more than 4 sample wells, largeror smaller sample wells, and even multiple waste reservoirs or otherchambers. Alternatively the waste reservoir can comprise a sponge orsimilar absorbent material. It can also comprise a vacuum system fluidlyattached to a waste reservoir as previously discussed, and of variousmaterials, with preference to inexpensive materials as discussedpreviously for the cartridge.

Variations of preferred embodiments of the apparatus for use with apreferred cartridge and sample tray combination as disclosed herein arealso possible within the scope of the present invention. An importantaspect of an alternative embodiment of the apparatus for use with apreferred embodiment of the cartridge and sample tray is thatembodiments of the apparatus are able to communicate with the cartridgeand sample tray, that the apparatus is equipped with appropriateelectronics and/or optics for a given, immunoassay, and that theapparatus is capable of performing or interfaced with an instrumentcapable of performing an automated immunoassay and of computingqualitative, semi-quantitative, and/or quantitative results. Anotheraspect of the apparatus for use with a preferred embodiment of thecartridge and sample tray combination is that the apparatus house orcontrol a suitably substantial portion of the means to move fluid in andout of the wells, capillary tubes, and chambers.

Aspects of an alternative embodiment of the apparatus for use withalternative embodiments of the cartridge and sample tray will varydepending on the design of alternative embodiments of the cartridge andsample tray, but preferably effectuate an immunoassay in capillarytubes, and is preferably relatively simple, rapid, reliable, andrequires minimal operator interaction.

Several variations of a preferred embodiment of the apparatus of thepresent invention can be employed for use with a fluorescenceimmunoassay or other immunoassays. A vacuum system can be employed inplace of a syringe within or in cooperation with the apparatus to removefluid from the capillary tubes and/or the sample tray. Similarly, themeans to mix the sample and reagent can be other than by magnetic fieldsuch as agitation by vibration or by physical mixing.

Various types of spectroscopic hardware can also be employed. Suchvariations are dictated by the immunoassay, analyte of interest, andother criteria and would be obvious to one of ordinary skill in the art.These variations include, but are not limited to, use of differentsignal generation means, including, but not limited to, argon lamps,xenon lamps, hydrogen lamps, deuterium lamps, tungsten lamps, nernstglower, nichrome wire, globar, and hollow cathode lamps or otherappropriate signal generation means capable of providing emitted signalscovering appropriate wavelengths in one or more regions of ultraviolet,visible, near infrared, infrared, and far infrared light; variouswavelength selectors including, but not limited to, filters, includinginterference filters and glass absorption filters, and monochromators,including prism monochromators, such as fluorite prism, fused silica orquartz prism, glass prism, sodium chloride prism, and potassium bromideprism; and gratings; and various signal detection means including, butnot limited to, photomultipliers, phototubes, photocells, silicondiodes, and semiconductors.

Variations to the signal processing means can also be employed toprovide quantitative results, such as constant automatic calibration ofthe signal generation and detection means, use of a more sophisticatedanalog to digital converter, use of multi-level calibration curves, e.g.a least squares fit of curves such as those shown in FIGS. 16a-d toFIGS. 17a-b, and data reduction and analysis that includes statisticalanalysis. The apparatus can also include a computer disc drive forloading a computer disc for storing a result provided by the signalprocessing or computing means in a computer storable file. The resultcan be transmitted via the RS-232 port on the apparatus to an externalcomputing or storage device for more sophisticated data reduction andanalysis. It should be noted that use of an erasable programmableread-only memory (EPROM) module, as in a preferred embodiment of theapparatus of the present invention, makes it relatively simple to modifythe software of the apparatus to adapt it to various immunoassayformats, levels of data reduction and analysis, and interaction withexternal and/or internal devices and/or components.

Alternative embodiments of the apparatus of the present invention canalso include various levels of automation. One embodiment of theapparatus of the present invention can be designed such that onlyinstrument control is possible with the stand alone apparatus and alldata reduction and analysis is performed by a computer connected ornetworked to the apparatus via a port, such as RS-232, IEEE-488 (HP-IB),contact closure, and the like. Similarly, control of the apparatus canbe automated by a connection to a computer as previously described whereall control inputs are directed by the computer and initiated by anoperator or alternatively by a computer program.

Additionally, a preferred embodiment of the apparatus of the presentinvention or variations of it can be interfaced with laboratory robots,such as cylindrical, cartesian, and articulated robots and the like; toenable complete automation of the immunoassay. The robotics device canbe programmed to remove sample from a central container and add it toeach well and alternatively to perform any pre-assay sample processingand preparation necessary, such as filtration, extraction, dilution,removal of certain components, and other manipulations depending on thesample. The robotics device can also place the sample tray and cartridgeon the apparatus and subsequently move the cartridge to the centrifugeor other device and dispose of the sample tray. The robotics device canbe integrated into an alternative embodiment of the apparatus of thepresent invention or it can be a commercially available robot for use inthe laboratory and known to those of ordinary skill in the art of thepresent invention.

EXAMPLES Example 1

Capillary-Tube Surface Preparation

The surface of borosilicate, glass capillary tubes (Drummond Scientific,Broomall, Pa.) was treated with a silanizing reagent in accordance withthe process for Aquasil® silanization coating in accordance with Example9, coating with a protein substrate conjugated to a reactive protein,blocking the capillary tubes, and drying and incubating. The lengths ofindividual capillary tubes were 3.5 centimeters (cm) with an innerdiameter of 0.65 millimeters (mm) and an outer diameter of 1 mm. Byusing these capillary tubes, one achieves a high surface area to volumeratio and minimizes the use of reagents. Other capillary tube dimensionscan be used. The high surface area to volume ratio allowed for a shorttwo-minute incubation. Tubes having other inner diameter dimensions werealso examined, however, the 0.65 mm inner diameter tubing was found tobe most useful with fresh, raw milk samples, which often contain fatglobules that can be as large as several hundred micrometers (μm). Suchfat particles can potentially clog the capillary tube channels if tubesof smaller inner diameter were used. Additionally, the transport ofreactants to the interior surface of the tube, where the measurablebinding reaction occurs, is strictly through diffusion. Therefore,potential problems due to irreproducible agitation are eliminated.

Example 2

Synthesis of Antigen Conjugates for Coating Capillary Tubes

The antigen conjugates, for coating onto the surface of the capillarytubes, were prepared by binding the appropriate β-lactam drug, such asPenicillin G, Ampicillin, Cloxacillin, Cephapirin, Ceftiofur,Amoxicillin and the like; to a carrier protein either bovine serumalbumin (BSA) or a polypeptide copolymer consisting of lysine andalanine subunits (Sigma Chemical Co., St. Louis, Mo.). The covalentlinking of antigen to carrier protein was accomplished through the useof conventional homobifunctional or heterobifunctional linkers, such asSuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),Bis(sulfosuccinimidyl) suberate, and1-Ethyl-3-(3-Dimethylaminopropyl)-carbodiimideHydrochloride (PierceChemical Co., Rockford, Ill.) in accordance with the process provided bythe manufacturer.

Example 3

General Procedure for Coating Antigen-Conjugate on the Surface ofCapillary Tubes

After the silanizing surface treatment, the capillary tubes wereincubated for from about 30 minutes to 24 hours in a buffered solutionof the antigen-conjugate (20-40 μg/ml). The incubation temperature wasusually 4-7° C., although occasionally room temperature incubation wasused. The capillary tubes were removed, washed with distilled water,dried in a stream of compressed air, and then incubated in a solution ofbovine-serum albumin (0.1% BSA in PBS-phosphate buffered saline-pH=7.2,0.05% Proclin 300 (Supelco) a biocide) for 1 hour at room temperature.The purpose of this incubation with BSA was to block any solutionregions of the surface that were not coated by the antigen-conjugate.The tubes were again removed, washed with distilled water and dried in astream of compressed air (20-30 psi). They were stored in the dark, atroom temperature in a sealed, foil pouch with an indicating desiccantpackage. Maintaining dry and dark conditions were important to maintainthe stability of the coated capillary tubes.

Example 4

Preparation of Fluorescent-labeled Antibody Conjugates

a. Antibody Production

Antibodies to the antibiotics in the penicillin family were produced byimmunizing goats with a conjugate of keyhole limpet hemocyanin (KLH)(Sigma Chemical) and ampicillin (Sigma). Ampicillin was used because ithas an amino-group available for conjugation. The resulting bleeds werescreened for cross-reactivity with penicillin G, ampicillin, cloxacillinand amoxicillin (Sigma). The cross-reactivity of this antibody withpenicillin G and ampicillin was comparable, while the cross-reactivityfor cloxacillin and amoxicillin was approximately 50% less. Thisantibody also exhibited less than 1% cross-reactivity with ceftiofur(UpJohn Inc.) and cephapirin (Sigma). Antibodies to ceftiofur weredeveloped by immunizing goats with KLH-ceftiofur. A monoclonal tocephapirin was developed using conventional techniques withKLH-cephapirin as the conjugate. Enzyme immunoassay studies indicatedthat the cross-reactivity of the ceftiofur antibody to the other fiveβ-lactam drugs of interest was less than 1%. The cross-reactivity of thecephapirin antibody to the other five β-lactam antibiotics was less than0.1%.

b. Fluorescently-Labeled Antibody Conjugate Production

Monoclonal and polyclonal antibodies were prepared using conventionalpurification techniques as taught by C. Schmidt, 1989, "The Purificationof Large Amounts of Monoclonal Antibodies," Journal of Biotechnology,Volume 11, pp 235-252. Using the fluorescent label Cy-5®, Cy-5®-antibodyconjugates were prepared using the protocols previously published andprovided by the manufacturer (Biological Detection Systems, Pittsburgh,Pa.). The Cy-5® fluorescent label, available with an NHS esterfunctionality, was linked to amino groups of the antibody. Purificationand isolation of the conjugated dye was performed throughchromatography. Spectroscopic examination indicated that the number ofCy-5® dye molecules that were bound to each antibody molecule wasusually between two and four. The ratio of antibody molecule to Cy-5®molecule is controlled by modifying reaction conditions such as time ofconjugation and/or ratio of Cy-5® concentration to antibodyconcentration in the reaction mixture. Batch to batch studies indicatedthat there was no significant increase in fluorescence intensity withincrease in the number of Cy-5® molecules per antibody molecule. Infact, the fluorescence intensity often decreased when thefluorophore/antibody ratio was greater than four. This phenomenon wasespecially apparent when using monoclonal antibodies. It is unclear atthis time whether this decrease was due to increased quenching with highloading of the fluorophore or due to decreased antibody binding affinityresulting from inactivation of the antibodies' recognition sites.

Example 5

Process for Analysis for Analytes in Samples

Immunoassay Protocol

(a) A measured amount of fluorescently-labeled conjugate, comprisingantibody-Cy-5® conjugate (preferably dried, e.g. lyophilized or airdried,) was combined with a sample of milk in a small container, such asthe well of a microtiter-plate. The raw milk sample and antibodyconjugate were mixed on a vibratory shaker for 10 seconds.

(b) The solution from (a) was then sipped into the appropriate capillarytube through the use of a manifold device, such as a modified pipettoror an embodiment of the cartridge of the present invention.

(c) The solution was then incubated, while in the capillary tube, for atime sufficient to react with the antigen attached to an interiorsurface of the tube, e.g. for 2 minutes.

(d) All reagents were then washed out of the capillary tubes withflowing distilled water from the end opposite from which the solutionwas brought in. The reagents were then dried with a stream of compressedair (20-30 psi).

(e) The fluorescence intensity of the tubes was then measured using afluorometer equipped with a 3 mW semiconductor-diode laser (λmax=635 nm,TOLD 9521 (s), Toshiba, Japan) signal generation means, appropriateoptics to filter the signal, and a current response of a silicon p-i-njunction diode to generate an analog photocurrent. The resultingphotocurrent was amplified, converted from an analog to a digitalsignal, and processed on a computer to determine the presence of analyteand semi-quantitatively measure an amount of analyte in a sample.

Processing via a computer involved comparing the fluorescence level ofthe detected signal with dose response curves as shown in FIG. 16a-b 16d; and FIG. 17a-17b. A normalized fluorescence signal is located on they-axis and the relative concentration in parts per billion was locatedon the x-axis. Table I shows U.S.F.D.A. safe/tolerance levels for thesix β-lactam drugs in milk.

Example 6

Preparation of a Capillary Tube for Use in a Competitive Immunoassay toDetect Cephapirin in Milk

A borosilicate glass capillary tube having a length of 65 mm and aninner diameter of 0.6 mm (Drummond Scientific) was washed with distilledwater and then dried under a stream of air until substantially all ofthe distilled water had evaporated from the surfaces of the capillarytube. The capillary tube was then incubated in a 2.5% aminopropyltriethoxysilate ethanol solution for 20 minutes at 80° C.

The incubated capillary tube was then washed with distilled water anddried in a stream of air. The dried capillary tube was then incubatedfor 2 hours at 120° C. After incubation, the capillary tube was cooledto room temperature.

The capillary tube was then incubated in the presence of a buffersolution comprising a conjugate of Cephapirin-Bovine Serum Albumin (BSA)conjugate. The cephapirin-BSA conjugate was prepared by combining 65 mgCephapirin, 46 mg EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide)and 26 mg of N-HS (N-hydroxysuccinimide)(Pierce Chemical) in a 16×100 mmglass tube. 1 ml DMF (N,N dimethylformamide) (Fisher Scientific) wasadded to the tube and the contents of the tube were stirred at roomtemperature for 30 minutes. 100 mg BSA was dissolved in 3 ml 20 mMpotassium phosphate buffer, pH 7.2, in a separate 16×100 glass tube. TheCephapirin solution was combined with the BSA solution and stirred atroom temperature for 1 hour. The Cephapirin-BSA conjugate was purifiedwith a gel filtration column (Sephadex G-25) using 20 mM potassiumphosphate buffer, pH 7.2, as the equilibration and elution buffer. Thecapillary tubes were incubated in the resultant Cephapirin-BSA conjugatefor 3 hours, during which time the Cephapirin-BSA conjugate became boundto the capillary tube surface. After incubation, the capillary waswashed with distilled water and dried under a stream of air.

The capillary tube was then incubated at 25° C. for 2 hrs in a blockingsolution of 10% sucrose, 0.1% bovine serum albumin and 0.05% proclin 300in order to block any free binding sites of the capillary tube surfacenot occupied by the antigen. After incubating the capillary tube in theblocking solution, the capillary tube was washed with distilled waterand dried under a stream of air.

Example 7

Preparation of Anti-Cephapirin-Cy-5® Conjugate

1 milligram of activated Cy-5® dye (Biological Detector Systems)dissolved in 0.2 ml of 20 mM potassium phosphate buffer, pH 7.2 wascombined with 2 ml of a Cephapirin antibody solution (4 mg/ml Cephapirinantibody in 20 mM potassium phosphate buffer at pH 7.2). Cy-5®dye/Cephapirin solution was then stirred at room temperature for 1 hour.The resultant Cy-5®-Cephapirin conjugate was purified on a gelfiltration column (Sephadex G-25), where 20 mM potassium phosphatebuffer, pH 7.2, was used as both the equilibration and elution buffer.

Example 8

Manual Immunoassay of Milk Suspected of Comprising Cephapirin

About 4 to 5 μl of antibody conjugate solution, as prepared in Example7, was added to 0.5 ml of milk suspected of comprising Cephapirin,resulting in an overall conjugate concentration in the milk of 10 μl/ml.The milk conjugate mixture was then incubated for a few seconds.

One end of the prepared capillary tube from Example 6 was dipped intothe incubated milk. A 10 μl plug of milk was taken up into the capillarytube under capillary force. Milk also coated the exterior of thecapillary tube up to about 20 mm.

The capillary tube was then turned upside down so that the 10 μl plug ofliquid moved down the capillary tube into a region in which the outersurface of the capillary tube had not contacted the milk. The sampleplug was incubated in the capillary tube for 1 minute to allow thereaction to proceed to provide a detectable fluorescent signal for apositive sample.

The plug was then washed from the capillary tube with 100 μl ofdistilled water. After washing the capillary tube, the capillary tubewas dried with a stream of air from an air gun.

The capillary tube was then irradiated with a laser, where thewavelength of light form the laser was 632.8 nm. Upon irradiation, ahighly intense emitted signal having a wavelength of 667 nm wasdetected. The intensity of the emitted signal indicated that nocephapirin was present in the assayed milk.

Example 9

Method for Coating 4-Amino-Penicillinic Acid on a Capillary Tube

AquaSil® Silanization Coating

One or more glass capillary tubes was immersed in a 0.2%AquaSil®--octodecyltriethoxy silane (Pierce Chemical #42797) deionizedwater solution in a manner that ensured a complete coating on thedesired portions of the capillary tubes and then incubated in thesolution for 40±30 minutes at room temperature. The capillary tube wasthen placed in the centrifuge and centrifuged at 1000 rpm for 10±5minutes to spin dry the capillary tube. Alternatively, the capillarytube may be air dried, dried with a gas, or an suitable means tosufficiently dry the capillary tube.

The AquaSil® coated capillary tube was then placed into a 125±20°C. ovenfor 60±10 minutes. Next, the capillary tube was allowed to cool to roomtemperature and subsequently stored in a desiccated, foil ziplock bag atroom temperature until further coating was required. The capillary tubeis stable for long periods in this storage condition.

BSA-Biotin Capillary Tube Coating

The AquaSil® coated capillary tube was immersed into a BSA-biotin(bovine serum albumin) solution consisting of a stock BSA-biotinsolution (Sigma #A-3294) at 200 μg/mL diluted 1:200 with 20 mM phosphatebuffered saline (PBS)-pH 7.2. 100 mL of the 20 mM PBS-pH 7.2 solutioncan be prepared by mixing 268 g potassium phosphate-dibasic (K₂ HPO₄)(Fisher #P284-3), 60.2 mg potassium phosphate-monobasic (KH₂ PO₄ H₂ O),and 875 mg sodium chloride (Fisher #P285-3) where the pH was 7.2±0.1.

After it was ensured that all desired areas were contacted withsolution, the BSA-biotin coated capillary tube was then incubated insolution for 1-24 hours at room temperature. After incubation, theBSA-biotin coated capillary tube was centrifuged at 1000 rpm for 10±5minutes to spin dry the capillary tube. The BSA-biotin coated capillarytube was then stored in a desiccated, foil ziplock bag at 4° C. untilfurther coating was required. The capillary tube was stable for longperiods in this storage condition.

Bovine Serum Albumin Blocking of Capillary Tubes

The BSA-biotin coated capillary tube was immersed in a 0.1% BSA/10%sucrose blocking solution. The 100 ml of the 0.1% BSA/10% sucroseblocking solution can be prepared by mixing 10 g sucrose (Fisher #S-53)with 80 mL deionized water, mixing the solution to dissolve the sucrose,adding 100 mg BSA (Sigma #A-3294) to the sucrose solution, slowly mixingthe sucrose-BSA solution, adding 0.05 mL of ProClin 300 (Supelco#4-8127), and adding additional deionized water to bring the volume to100 mL.

After it was ensured that all desired areas were contacted theBSA-Sucrose blocked capillary tube was then incubated for 1-24 hours atroom temperature. After incubation the capillary tube was centrifuged at1000 rpm for 10±5 minutes to spin dry the capillary tube.

The BSA-biotin and BSA-Sucrose coated capillary tube was then stored ina desiccated, foil ziplock bag at 4° C. until further coating wasrequired. The capillary tube is stable for long periods at thiscondition.

NeutrAvidin-4-Amino-Penicillinic Acid Capillary Coating

The BSA-biotin/BSA-Sucrose coated capillary tube was immersed in asolution of 40 μg/mL neutravidin-amino-penicillinic acid (NAV-APA)conjugate, which NAV-APA conjugate solution was prepared by dilutingstock NAV-APA conjugate solution diluted to 40 g/mL with 20 mM PBS-pH7.2.

After it was ensured that all the desired portions of the capillarytubes were contacted with solution the NAV-APA coated capillary tube wasthen incubated for 1-24 hours at room temperature. After incubation, theNAV-APA coated capillary tube was centrifuged at 1000 rpm for 10±5minutes to spin dry the capillary tube.

The NAV-APA coated capillary tube was then immersed in a containercontaining deionized water such that all surfaces of the capillary tubewere contacted by the water. The NAV-APA coated capillary tube was thencentrifuged at 1000 rpm for 10±5 minutes to spin dry the capillary tube.

The NAV-APA coated capillary tube was then placed in a vacuum ovenheated to 37° C. The oven was evacuated to 24 in. Hg. The coatedcapillary tube was heated under vacuum for 60 minutes. The oven was thenturned off, the vacuum line closed, and vacuum pump turned off. TheNAV-APA coated capillary tube was then kept in the evacuated oven overnight.

The AquaSil®/BSA-biotin/NAV-APA coated capillary tube can be kept withthe baffled insert in a desiccated, foil ziplock bag or the capillarytube can be decanted into a sealable, desiccated container and placed ina foil ziplock bag and stored at 4° C. until ready for use in a manualversion of the immunoassay or for assembly of the cartridges of thepresent invention.

Example 10

Method for Coating a Ceftiofur Capillary Tube

One or more capillary tubes was prepared with the Aquasil® capillarycoating procedure, the BSA-Biotin capillary coating procedure, and theBSA blocking procedure as described in Example 9.

The BSA-biotin and BSA-Sucrose coated capillary tube was immersed in a20 μg/mL neutravidin-ceftiofur conjugate solution such that all desiredportions of the capillary tube were contacted by the conjugate solution,which conjugate solution was prepared by diluting stock NAV-Ceftiofurconjugate solution to 20 μg/mL in 20 mM PBS-pH 7.2.

The NAV-Ceftiofur coated capillary tube was then incubated for 1-24hours at room temperature. After incubation, the NAV-Ceftiofur coatedcapillary tube was centrifuged at 1000 rpm for 10±5 minutes to spin drythe capillary tube.

The NAV-Ceftiofur coated capillary tube was then immersed in a containercontaining deionized water such that all desired portions of thecapillary tube were contacted with the deionized water.

The NAV-Ceftiofur coated capillary tube was then centrifuged at 1000 rpmfor 10±5 minutes to spin dry the capillary tube.

The NAV-Ceftiofur coated capillary tube was then placed in a vacuum ovenheated to 37° C. The oven was evacuated to 25 in. Hg. The coatedcapillary tube was heated under vacuum for 60 minutes. The oven was thenturned off, the vacuum line closed, and vacuum pump turned off. TheNAV-Ceftiofur coated capillary tube was then kept in the evacuated ovenover night.

The AquaSil/BSA-biotin/NAV-Ceftiofur coated capillary tube can be keptwith the baffled insert in a desiccated, foil ziplock bag or thecapillary tube can be decanted into a sealable, desiccated container andplaced in a foil ziplock bag and stored at 4° C. until ready for use ina manual version of the immunoassay or for assembly of the cartridges ofthe present invention.

Example 11

Method for Coating Cephapirin Capillary Tubes

One or more glass capillary tubes was prepared with the AquaSil coatingprocedure of Example 9. The AquaSil coated capillary tube was thenimmersed in a 25 μg/mL BSA-Cephapirin coating solution such that alldesired portions of the capillary tube were contacted with the coatingsolution, which BSA-Cephapirin coating solution was prepared by dilutingstock BSA-Cephapirin conjugate solution to 25 μg/mL with 20 mM PBS-pH7.2.

The BSA-Cephapirin coated capillary tube was then incubated for 1-24hours at room temperature. After incubation the capillary tube wascentrifuged at 1000 rpm for 10±5 minutes to spin dry the capillary tube.

The BSA-Cephapirin coated capillary tube was then stored in adesiccated, foil ziplock bag at 4° C. until further coating wasrequired. The capillary tube is stable for long periods at thiscondition.

The BSA-Cephapirin coated capillary tube was then coated with the BSAblocking solution described in Example 9. The BSA-Cephapirin coated andBSA-sucrose blocked capillary tube was then immersed in a containercontaining deionized water such that all desired portions of thecapillary tube were contacted with the water.

The BSA-Cephapirin coated and BSA-sucrose blocked coated capillary tubewas then centrifuged at 1000 rpm for 10±5 minutes to spin dry thecapillary tube.

The BSA-Cephapirin coated and BSA-sucrose blocked capillary tube wasthen placed in a vacuum oven heated to 37° C. The oven was evacuated to25 in. Hg. The coated capillary tube was heated under vacuum for 60minutes. The oven was then turned off, the vacuum line closed, andvacuum pump turned off. The NAV-Ceftiofur coated capillary tube was thenkept in the evacuated oven over night.

The AquaSil®/BSA-Cephapirin/BSA-sucrose coated capillary tube can bekept with the baffled insert in a desiccated, foil ziplock bag or thecapillary tube can be decanted into a sealable, desiccated container andplaced in a foil ziplock bag and stored at 4° C. until ready for use ina manual version of the immunoassay or for assembly of the cartridges ofthe present invention.

Example 12

Method of the immunoassay Used With the Devices and Apparatus of thePresent Invention

The immunoassay was begun by placing preferred embodiments of thecartridge and sample tray on a preferred embodiment of the apparatus ofthe present invention (also called the Parallux Processor Idetek,Sunnyvale, Calif.) and adding 100 microliters of raw, unpasteurized milkinto each well of the sample tray. A barcode with calibrationinformation for the particular kit was scanned. Appropriateidentification information was entered via the keypad, and the apparatusbegan running the immunoassay. Movement of magnets by the magnetic mixerunder the tray agitated the metal washers in the sample tray-wells for20 seconds so the dry reagent comprising a fluorescent label in eachwell was homogeneously dissolved in the sample milk. After mixing, thesample tray was raised by the lift motor so that the tips of thecapillary tubes in the cartridge were immersed in the milk. A syringepump was actuated to draw milk into the capillary tubes, where itremained for an incubation period of two minutes. Upon completion ofincubation, the syringe pump was used to wash out all contents of thetubes into the tray. The wash solution came from a reservoir in theapparatus. The sample tray was discarded, and the cartridge was placedon the centrifuge (called a read station). The centrifuge spun at 3000rpm for 20 seconds to eject any remaining wash solution from the tubes.This step was followed by exposure to a diode laser beam for measurementof fluorescence. The fluorescence signals from each tube were collected,amplified, converted to a digital signal and processed.

The cartridge used in this Example contained reagents for three separateimmunoassays. The fourth tube and corresponding well were blank. Thefirst immunoassay was for cephapirin, the second for ceftiofur, and thethird for the penicillin family of drugs (penicillin G, ampicillin,cloxacillin, and amoxicillin). Therefore, a single cartridge was able toscreen for six analytes.

For semi-quantitative immunoassays, the fluorescence signals werecompared to a value that had been input by the barcode. Any measuredfluorescence value above the barcoded value resulted in a pass, and anymeasured value equal to or below the barcoded value resulted in a fail.

For quantitative immunoassays, the raw fluorescence signals were plottedas a function of spiked concentration in raw milk to produce amulti-level standard curve.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationscan be practiced within the scope of the appended claims. Therefore, thespirit and scope of the appended claims should not be limited to thedescription of preferred versions contained herein

What is claimed is:
 1. A process for screening for an analyte in asample, which process comprisesimporting a fluid mixture into acapillary tube having a wall and coated on at least a portion of itsinterior surface with a substrate, wherein the fluid mixture comprises asample suspected of containing the analyte and a reagent comprising afluorescently-labeled conjugate that(a) binds to the analyte or to theanalyte and the substrate and (b) fluoresces when irradiated with anappropriate electromagnetic signal; maintaining the fluid mixture in thecapillary tube for a time sufficient for binding to take place betweenthe substrate and the fluorescently-labeled conjugate; removing excessfluid mixture from the capillary tube; externally irradiating the coatedportion of the capillary tube with an electromagnetic signal sufficientto cause fluorescence of bound fluorescently labeled conjugate; anddetecting through the capillary tube wall the resulting fluorescenceoccurring on the capillary tube interior surface to screen for theanalyte.
 2. The process of claim 1, wherein the capillary tube is driedafter the removal of excess fluid from the capillary tube and prior todetecting the resultant fluorescence.
 3. The process of claim 2, whereinthe capillary tube is dried by spinning the capillary tube on acentrifuge, which is designed to securely hold the capillary tube andspinning it for a sufficient time and at a sufficient speed to dry thecapillary tube.
 4. The process of claim 3, wherein the capillary tube isheld by a cartridge designed to fit and be securely held on saidcentrifuge.
 5. The process of claim 2, wherein the means to dry thecapillary tube comprises aspirating the capillary tube with a stream ofgas.
 6. The process of claim 1, wherein the binding in the capillarytube involves binding a fluorescently-labeled conjugate to the substrateof the interior surface of the capillary tube.
 7. The process of claim6, wherein the electromagnetic signal is generated by a laser or atungsten lamp.
 8. The process of claim 1, wherein the detectedfluorescence is used to determine the amount of analyte present.
 9. Theprocess of claim 1, wherein data reduction and data analysis of detectedfluorescence is used to give a result.
 10. The process of claim 9,wherein the result of the process is presented as a digital display, aprintout, a computer storable file, an output to an external device, orany combination of the foregoing.
 11. The process of claim 8, whereindata reduction and data analysis of detected fluorescence is used todetermine the amount of analyte present.